Method for Generating Aptamers with Improved Off-Rates

ABSTRACT

The present disclosure describes improved SELEX methods for producing aptamers that are capable of binding to target molecules and improved photoSELEX methods for producing photoreactive aptamers that are capable of both binding and covalently crosslinking to target molecules. Specifically, the present disclosure describes methods for producing aptamers and photoaptamers having slower dissociation rate constants than are obtained using prior SELEX and photoSELEX methods. The disclosure further describes aptamers and photoaptamers having slower dissociation rate constants than those obtained using prior methods. In addition, the disclosure describes aptamer constructs that include a variety of functionalities, including a cleavable element, a detection element, and a capture or immobilization element.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/414,026, filed May 16, 2019, which is a continuation of U.S.application Ser. No. 15/878,161, filed Jan. 23, 2018, which issued asU.S. Pat. No. 10,316,321 on Jun. 11, 2019, which is a continuation ofU.S. application Ser. No. 15/410,414, filed Jan. 19, 2017, nowabandoned, which is a continuation of U.S. application Ser. No.14/610,758, filed Jan. 30, 2015, now abandoned, which is a continuationof U.S. application Ser. No. 13/113,261, filed May 23, 2011, whichissued as U.S. Pat. No. 8,975,388 on Mar. 10, 2015, which is adivisional of U.S. application Ser. No. 12/175,434, filed Jul. 17, 2008,which issued as U.S. Pat. No. 7,947,447 on May 24, 2011, which claimsthe benefit of U.S. Provisional Application Ser. No. 60/950,281, filedJul. 17, 2007, U.S. Provisional Application Ser. No. 60/950,293, filedJul. 17, 2007, U.S. Provisional Application Ser. No. 60/950,283, filedJul. 17, 2007, U.S. Provisional Application Ser. No. 61/031,420, filedFeb. 26, 2008 and U.S. Provisional Application Ser. No. 61/051,594,filed May 8, 2008. U.S. application Ser. No. 12/175,434 is also acontinuation in part of U.S. application Ser. No. 11/623,580 and U.S.application Ser. No. 11/623,535, each of which was filed on Jan. 16,2007, and each of which are now abandoned. Each of these applicationsare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to methods for the generationof aptamers and photoaptamers having improved properties and theimproved aptamers and photoaptamers generated thereby. In particular,the present disclosure describes slow off-rate aptamers that are highlyspecific to a target of interest. The disclosure describes thecomposition of these slow off-rate aptamers as well methods for theirselection. Further the disclosure describes aptamer constructs withimproved functionalities for detection methods. Further, the disclosuredescribes applications enabled by these improved aptamers.

SEQUENCE LISTING

Incorporated by reference herein in its entirety is the Sequence Listingentitled “sml_19D_sequence_ST25.txt”, created Jul. 28, 2018, size of 6kilobytes.

BACKGROUND

The following description provides a summary of information relevant tothe present disclosure and is not a concession that any of theinformation provided or publications referenced herein is prior art tothe claimed invention.

The SELEX process is a method for the in vitro selection of nucleic acidmolecules that are able to bind with high specificity to targetmolecules and is described in U.S. Pat. No. 5,475,096 entitled “NucleicAcid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813)entitled “Nucleic Acid Ligands” each of which is specificallyincorporated by reference herein. These patents, collectively referredto herein as the SELEX Patents, describe methods for making an aptamerto any desired target molecule.

The basic SELEX process has been modified to achieve a number ofspecific objectives. For example, U.S. Pat. No. 5,707,796, entitled“Method for Selecting Nucleic Acids on the Basis of Structure” describesthe use of the SELEX process in conjunction with gel electrophoresis toselect nucleic acid molecules with specific structural characteristics,such as bent DNA. U.S. Pat. No. 5,580,737, entitled “High-AffinityNucleic Acid Ligands That Discriminate Between Theophylline andCaffeine” describes a method for identifying highly specific aptamersable to discriminate between closely related molecules, termedCounter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Solution SELEX” describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev” describes methods for obtaining improved aptamers after SELEXhas been performed. U.S. Pat. No. 5,705,337, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chemi-SELEX” describesmethods for covalently linking an aptamer to its target.

The SELEX process encompasses the identification of high-affinityaptamers containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides” thatdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe).

Further modifications of the SELEX process are described in U.S. Pat.Nos. 5,763,177, 6,001,577, and 6,291,184, each of which is entitled“Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX”; see also,e.g., U.S. Pat. No. 6,458,539, entitled “Photoselection of Nucleic AcidLigands”. These patents, collectively referred to herein as “thePhotoSELEX Patents” describe various SELEX methods for selectingaptamers containing photoreactive functional groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.The resulting photoreactive aptamers are referred to asphotocrosslinking aptamers or photoaptamers.

Although these SELEX and photoSELEX processes are useful, there isalways a need for processes that lead to improved properties of aptamersgenerated from in vitro selection techniques. For example, a need existsfor aptamers to target molecules with better binding affinities thanthose achieved with naturally occurring DNA or RNA nucleotides, as wellas methods for producing such aptamers. For many applications, such asfor example, in vitro assays, diagnostics, therapeutic, or imagingapplications, it is of interest to produce aptamers with slowdissociation rates from the aptamer/target affinity complex. Severaltechniques have been proposed for producing such reagents (see, e.g., WO99/27133 and US 2005/0003362). However, these selection processes do notdiscriminate between the selection of reagents that have fastassociation kinetics with the target (i.e., fast on-rates) and theselection of reagents that have slow dissociation kinetics with thetarget (i.e., slow off-rates). Thus, there is a need for novel processesand techniques that favor the selection of slow off-rate aptamers whileinhibiting the selection of aptamers that simply have a fast associationrate with the target.

Finally, there is a need for aptamer constructs that include differentbuilt-in functionalities. These functionalities may include tags forimmobilization, labels for detection, means to promote or controlseparation, etc.

SUMMARY

The present disclosure describes novel aptamers, and methods to produceand use such aptamers. In particular, the disclosure describes slowoff-rate (slow rate of dissociation) aptamers, slow off-rate aptamerscontaining C-5 modified pyrimidines, and processes for the selection ofslow off-rate aptamers by dilution, by the addition of a competitor, orby a combination of both approaches. In addition, slow off-rate aptamersto various targets such as proteins and peptides are described. Slowoff-rate aptamers with unique structural features and meltingtemperatures are also described. The disclosure also describes slowoff-rate aptamers with photoreactive functional groups, aptamers thatare refractory to the presence of poly-anionic materials, and aselection process for these aptamers, as well as aptamers constructedwith a variety of other functionalities to improve their utility invarious applications.

The present disclosure describes improved SELEX methods for generatingaptamers that are capable of binding to target molecules. Morespecifically, the present disclosure describes methods for producingaptamers and/or photoaptamers having slower rates of dissociation fromtheir respective target molecules than aptamers and photoaptamersobtained with previous SELEX methods. Generally, after contacting thecandidate mixture with the target molecule and allowing the formation ofnucleic acid-target complexes to occur, a slow off-rate enrichmentprocess is introduced wherein nucleic acid-target complexes with fastdissociation rates will dissociate and not reform, while complexes withslow dissociation rates will remain intact. Methods for introducing aslow off-rate enrichment process include, but are not limited to, addingcompetitor molecules to the mixture of nucleic acids and targetmolecules, diluting the mixture of nucleic acids and target molecules,or a combination of both of these. The disclosure further describesaptamers and photoaptamers obtained using these methods.

In one embodiment, the method comprises preparing a candidate mixture ofnucleic acids; contacting the candidate mixture with a target moleculewherein nucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; introducing a slow off-rate enrichmentprocess to induce the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; partitioning theremaining bound nucleic acid-target molecule complexes from free nucleicacids in the candidate mixture; and identifying the nucleic acids thatwere bound to the target molecule. The process may further include theiterative step of amplifying the nucleic acids that bind to the targetmolecule to yield a mixture of nucleic acids enriched with nucleic acidsthat bind to the target molecule yet produce nucleic acid-targetmolecule complexes having slow dissociation rates.

In another embodiment, the candidate mixture of nucleic acids includesnucleic acids containing modified nucleotide bases that may aid in theformation of modified nucleic acid-target complexes having slowdissociation rates. Improved methods for performing SELEX with modifiednucleotides, including nucleotides which contain photoactive or otherfunctional groups, or nucleotides which contain placeholders forphotoactive groups are disclosed in U.S. application Ser. No.12/175,388, filed Jul. 17, 2008, which is incorporated by referenceherein in its entirety, and entitled “Improved SELEX and PHOTOSELEX”which is being filed concurrently with the instant application.Placeholder nucleotides may also be used for the mid-SELEX or post-SELEXintroduction of modified nucleotides that are not photoreactive.

The various methods and steps described herein can be used to generatean aptamer capable of either (1) binding to a target molecule or (2)binding to a target molecule and subsequently forming a covalent linkagewith the target molecule upon irradiation with light in the UV orvisible spectrum.

In another aspect, the various methods and steps described herein can beused to generate an aptamer capable of modifying the bioactivity of atarget molecule through binding and/or crosslinking to the targetmolecule. In one embodiment, an aptamer to a unique target moleculeassociated with or relevant to a specific disease process is identified.This aptamer can be used as a diagnostic reagent, either in vitro or invivo. In another embodiment, an aptamer to a target molecule associatedwith a disease state may be administered to an individual and used totreat the disease in vivo. The aptamers and photoaptamers identifiedherein can be used in any diagnostic, imaging, high throughput screeningor target validation techniques or procedures or assays for whichaptamers, oligonucleotides, antibodies and ligands, without limitationcan be used. For example, aptamers and photoaptamers identified hereincan be used according to the methods described in detail in theconcurrently filed U.S. application Ser. No. 12/175,446, entitled“Multiplexed Analyses of Test Samples”, which is incorporated byreference herein in its entirety.

Previous aptamers that do not have the slow off-rate properties of theaptamers of the present invention have been used for a variety ofpurposes. In almost all such uses, slow off-rate aptamers will haveimproved performance relative to aptamers not selected to have slowoff-rate properties.

The aptamer Macugen®, (See, e.g., U.S. Pat. Nos. 6,168,778; 6,051,698;6,426,335; and 6,962,784; each of which is incorporated herein byreference in its entirety) has been approved for the treatment ofmacular degeneration, and functions due to its specific affinity forVEGF. Other aptamers have been studied and/or are in development for useas therapeutic agents. Aptamers not selected to have slow off-rateproperties have also been used in many diagnostic and imagingapplications (See, e.g., U.S. Pat. Nos. 5,843,653; 5,789,163; 5,853,984;5,874,218; 6,261,783; 5,989,823; 6,177,555; 6,531,286; each of which isincorporated herein by reference in its entirety), high-thorough putscreening (See, e.g., U.S. Pat. Nos. 6,329,145; 6,670,132; 7,258,980;each of which is incorporated herein by reference in its entirety) andin PCR kits (See, e.g., U.S. Pat. Nos. 6,183,967; 6,020,130; 5,763,173;5,874,557; 5,693,502; each of which is incorporated herein by referencein its entirety.) The slow off-rate aptamers of this disclosure may beused in any diagnostic, therapeutic, imaging or any other use for whichantibodies, aptamers and ligand binding pairs have been used.

In another aspect, the disclosure provides aptamers and photoaptamersidentified by the improved methods disclosed herein, diagnostic kitsthat include such aptamers and photoaptamers, and therapeutic anddiagnostic uses of such aptamers and photoaptamers. The novel, slowoff-rate aptamers and photoaptamers identified using the describedmethods can be used in a variety of assays including, assays that useplanar arrays, beads, and other types of solid supports. The assays maybe used in a variety of contexts including in life science researchapplications, clinical diagnostic applications, (e.g., a diagnostic testfor a disease, or a “wellness” test for preventative healthcare); ALONAand UPS assays, and in vivo imaging applications. For some applications,multiplexed assays employing the described aptamers and photoaptamersmay be used.

In some embodiments, the slow off-rate aptamers (or photoaptamers)described herein can be used as intravenous or oral contrast agents forCAT scans and other imaging applications. CAT scans are used in thediagnosis of muscle and bone disorders, locating blood clots, detectinginternal bleeding, monitoring diseases such as cancer, etc. The slowoff-rate aptamers may be labeled with a CAT scan detectable component,such as, for example, iodine, barium, or gastrograffin. In addition tocarrying the detectable component, the aptamer may be designed to directthat component to a specific tissue or desired target. The aptamer mayserve to concentrate or localize the detectable component and thusimprove the signal to noise ratio by increasing available signal.Because the off-rate of the aptamer can be sufficiently slow, theduration of the scan can be increased, and the signal to noise ratio ofthe scan may be improved. The specificity of the aptamer for the targetmay also improve the signal to noise ratio in these imagingapplications.

In one embodiment, the slow off-rate aptamer is labeled with adiamagnetic or paramagnetic material. In this embodiment, the labeledaptamer may be used to improve the performance of magnetic resonanceimaging (MM). MRI is particularly well suited to the imaging of small,selective areas and tissues with high water content or to monitoringblood flow. The specificity of the slow off-rate aptamers may improvethe localization of the MM reagent to a desired tissue section.Similarly, slow off-rate aptamers may be modified with materials such asfluorine, carbon11, oxygen15, or nitrogen13, for use in PET scans. Inanother embodiment, the aptamers may be labeled with IR active materialsthat may be used for infrared imaging. It is also contemplated that slowoff-rate aptamers may be labeled for use with other imaging modalities.

In one embodiment, the slow off-rate aptamers may be used as verysensitive and specific reagents for incorporation into a variety of invitro diagnostic methods or kits. In some embodiments, the slow off-rateaptamers are used as substitutes for antibodies in a number ofinfectious, or other type of, disease detection methods where theaptamer to the target of interest includes either or both a detectablematerial and an immobilization or capture component. In theseembodiments, after the aptamer from the kit is mixed with a clinicalspecimen, a variety of assay formats may be utilized. In one embodiment,the aptamer also includes a detectable label, such as a fluorophore. Inother embodiments, the assay format may include fluorescence quenching,hybridization methods, flow cytometry, mass spectroscopy, inhibition orcompetition methods, enzyme linked oligonucleotide assays, SPR,evanescent wave methods, etc. In some embodiments, the aptamer isprovided in the kit in solution. In other embodiments, the aptamer inthe kit is immobilized onto a solid support used in conjunction with theassay for testing the specimen. In various embodiments, the solidsupport is designed for the detection of one or more targets ofinterest. In other embodiments, the kit may further include reagents toextract the target of interest, reagents for amplifying the aptamer,reagents for performing washing, detection reagents, etc.

In another embodiment, the slow off-rate aptamers may be used intherapeutic imaging studies. During the development of new therapeuticcompounds, it is often difficult to assess certain characteristics ofthe compound, such as, for example, biodistribution, the washout rate,bioavailability, in vivo drug/target interactions, etc. In many cases,if a suitable detectable material was used to modify the therapeuticcompound, imaging studies could be used to assess all of thesecharacteristics. Though direct modification of a therapeutic compoundfrequently inhibits its ability to interact with its target and thusreduces efficacy, an aptamer's small size and customizable specificity,render it potentially well-suited to react with a therapeutic compound(for example, an antibody or other protein-based therapeutic) whileminimizing any undesirable effects on the compound's therapeuticefficacy. To assess such characteristics as biodistribution and thewashout rate, the aptamer/therapeutic complex may survive for anextended period of time. These types of studies may be simplified incases where the therapeutic compound is a slow off-rate aptamer. Invarious embodiments, aptamers used in therapeutic, imaging, anddiagnostic applications may include various modifications, such as, forexample, 2′ fluoro and other modifications, to increase the stability ofthe aptamer upon exposure to various components that may be present in atest sample or in vivo, such as, for example, nucleases and other sampleor bodily fluid components.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates an exemplary SELEX method and FIG. 1B illustrates anexemplary SELEX method which includes the step of incorporating a slowoff-rate enrichment process or process.

FIG. 2 illustrates representative aptamer template, primer, andcomplementary oligonucleotide sequences used in the disclosure. Theoligonucleotides were prepared by standard solid-phase synthesistechniques. B=dT-biotin.

FIGS. 3A and 3B illustrate histograms of dissociation rate constants foraffinity aptamers selected without (FIG. 3A) and with (FIG. 3B) a slowoff-rate enrichment process as described in Example 2.

FIGS. 4A and B show oligonucleotides that were used to prepare thecandidate mixtures or perform various steps in the selection processdescribed in Example 2. The oligonucleotides were prepared by standardsolid-phase synthesis techniques. BrdU (5-bromo-dUTP), Anthraquinone(AQ), and psoralen (Psor) chromophores were purchased asphosphoramidites and added to the 5′ terminus of the forward primerduring synthesis. 4-azido-2-nitro-aniline (ANA) was prepared as apara-nitro-phenyl carbonate derivative and coupled to a 5′ hexylaminephosphoramidite after synthesis. Two candidate mixture sequences wereused in this example, designated 1 and 2. B=dT-biotin. (FIG. 4A)Template 1 was only used with candidate mixtures containing 5′-BrdU, AQ,and ANA, and (FIG. 4B) Template 2 was only used with candidate mixturescontaining 5′-Psor for Example 2.

FIG. 5 illustrates chemical structures of the chromophores coupled tothe 5′ terminus of the forward primer as illustrated in FIGS. 4A and 4B.

FIG. 6 illustrates a PAGE analysis of crosslink activity of TIMP-35′ANA/BndU enriched library using 5′-Fixed PhotoSELEX described inExample 3. The gel illustrates the separation of free aptamer (A_(f)),intramolecular crosslinked aptamer (A_(f)*), and crosslinkedprotein:aptamer complexes (P:A).

FIG. 7 is a chart of over 500 targets for which aptamers have beenidentified. Many of these aptamers have been designed to have slowdissociation rates from their respective targets.

FIGS. 8A to 8D illustrate aptamer constructs that contain a variety ofdifferent and optional functionalities including immobilization tags,labels, photocrosslinking moieties, spacers, and releasable moieties.

FIGS. 9 A to 9F illustrate examples of aptamer constructs including acleavable or releasable element, a tag (for example biotin), a spacer,and a label (for example Cy3).

FIG. 10 illustrates the aptamer and primer constructs described in thedisclosure. Cy3 represents a Cyanine 3 dye, PC a photocleavable linker,ANA a photoreactive crosslinking group, (AB)₂ a pair of biotin residuesseparated by dA residues, and (T)₈ a poly dT linker. Primer constructsare complementary to the complete 3′ fixed region of the aptamerconstructs.

FIGS. 11 A to 11 C illustrate dose response curves for slow off-rateaptamers versus traditional aptamers for three different targets.

FIGS. 12 A and 12B illustrate performance curves for a slow off-rateaptamer where the target was a peptide.

FIG. 13 illustrates a plot of the measured melting temperature of anumber of slow off-rate aptamers relative to the predicted meltingtemperature.

FIG. 14 describes the base modifications of nucleotides included in thisdisclosure. The R groups that may be used are described in addition tothe linkers (X) that may be used between the nucleotide attachment pointand the R group. The positions of attachment for the various “R” groupsare also indicated on the respective R group.

FIG. 15 illustrates a plot used in the determination of the bindingconstant for a slow off-rate aptamer containing C-5 modified pyrimidinesto thrombin.

DETAILED DESCRIPTION

The practice of the invention disclosed herein employs, unless otherwiseindicated, conventional methods of chemistry, microbiology, molecularbiology, and recombinant DNA techniques within the level of skill in theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition).

All publications, published patent documents, and patent applicationscited in this specification are indicative of the level of skill in theart(s) to which the invention pertains. All publications, publishedpatent documents, and patent applications cited herein are herebyincorporated by reference to the same extent as though each individualpublication, published patent document, or patent application wasspecifically and individually indicated as being incorporated byreference.

As used in this specification, including the appended claims, thesingular forms “a,” “an,” and “the” include plural references, unlessthe content clearly dictates otherwise, and are used interchangeablywith “at least one” and “one or more.” Thus, reference to “an aptamer”includes mixtures of aptamers, reference to “a probe” includes mixturesof probes, and the like.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical values such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

As used herein, “nucleic acid ligand” “aptamer” and “clone” are usedinterchangeably to refer to a non-naturally occurring nucleic acid thathas or may have a desirable action on a target molecule. A desirableaction includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waythat modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.In one embodiment, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure, other than a polynucleotide, that binds to the aptamerthrough a mechanism which is predominantly independent of Watson/Crickbase pairing or triple helix binding, wherein the aptamer is not anucleic acid having the known physiological function of being bound bythe target molecule. Aptamers include nucleic acids that are identifiedfrom a candidate mixture of nucleic acids, the aptamer being a ligand ofa given target, by the method comprising: (a) contacting the candidatemixture with the target, wherein nucleic acids having an increasedaffinity to the target relative to other nucleic acids in the candidatemixture may be partitioned from the remainder of the candidate mixture;(b) partitioning the increased affinity and/or slow off-rate nucleicacids from the remainder of the candidate mixture; and (c) amplifyingthe increased affinity, slow off-rate nucleic acids to yield aligand-enriched mixture of nucleic acids, whereby aptamers to the targetmolecule are identified. It is recognized that affinity interactions area matter of degree; however, in this context, the “specific bindingaffinity” of an aptamer for its target means that the aptamer binds toits target generally with a much higher degree of affinity than it maybinds to other, non-target, components in a mixture or sample. An“aptamer” or “nucleic acid ligand” is a set of copies of one type orspecies of nucleic acid molecule that has a particular nucleotidesequence. An aptamer can include any suitable number of nucleotides.“Aptamers” refer to more than one such set of molecules. Differentaptamers may have either the same number or a different number ofnucleotides. Aptamers may be DNA or RNA and maybe single stranded,double stranded, or contain double stranded regions.

As used herein, “slow off-rate” or “slow rate of dissociation” or “slowdissociation rate” refers to the time it takes for an aptamers/targetcomplex to begin to dissociate. This can be expressed as a half life,t_(1/2), or the point at which 50% of the aptamer/target complex hasdissociated. The off-rate or dissociation rate of a slow off-rateaptamer, expressed as t_(1/2) values, can be about ≥30 min., ≥about 60min., ≥about 90 min., ≥about 120 min.≥about 150 min.≥about 180min.≥about 210 min., and ≥about 240 min.

In one embodiment, a method for producing a synthetic library of nucleicacids comprises: 1) synthesizing the nucleic acids; 2) deprotecting thenucleic acids; 3) purifying the nucleic acids; and 4) analyzing thenucleic acids. In the synthesis step, a monomer mixture is preparedwhere the ratio of the various nucleotides in the mix is optimized toyield equal ratios of each nucleotide in the final product. One or moreof the monomers in the mixture may comprise a modified nucleotide.Amidite protection groups are used in this procedure and in oneembodiment, the monomer concentration is 0.1M. During synthesis, thefive prime protecting group is retained in the product nucleic acid.Synthesis is conducted on a solid support (controlled pore glass, CPG)and at least about 80 cycles are completed to synthesize the finalproduct.

After the synthesis process, the nucleic acid product is deprotected. A1.0 M aqueous lysine buffer, pH 9.0 is employed to cleave apurinic siteswhile the product is retained on the support (controlled pore glass,CPG). These cleaved truncated sequences are washed away with deionized(dI) water two times. 500 uL of dI water are added after the two washesin preparation for the deprotection step. This step involves thetreatment with 1.0 mL of t-butylamine:methanol:water, 1:1:2, for 5 hoursat 70° C., is followed by freezing, filtration, and evaporation todryness. The nucleic acid product is purified based on thehydrophobicity of the protecting group on a PRP-3 HPLC column(Hamilton). Appropriate column fractions are collected and pooled,desalted, and evaporated to dryness to remove the volatile elutionbuffers. The final product is washed with water by a centrifugationprocess and then re-suspended. Finally the resuspended material istreated to deprotect the final product. Final product is characterizedby base composition, primer extension, and sequencing gel.

A candidate mixture of nucleic acids, or a library of nucleic acids, mayalso be produced by an enzymatic method using a solid phase. In oneembodiment, this method comprises the same basic steps described above.In this case the goal is the synthesis of an antisense library and theselibraries are produced with a 5′ biotin modification. All remainingsynthetic processes are as described above. Once the synthetic libraryis prepared, the nucleic acids maybe used in a primer extension mixcontaining one or more modified nucleotides to produce the finalcandidate mixture in a classic primer extension method.

Aptamers may be synthesized by the same chemistry that is used for thesynthesis of a library. However, instead of a mixture of nucleotides,one nucleotide is introduced at each step in the synthesis to controlthe final sequence generated by routine methods. Modified nucleotidesmay be introduced into the synthesis process at the desired positions inthe sequence. Other functionalities may be introduced as desired usingknown chemical modifications of nucleotides.

As used herein, “candidate mixture” is a mixture of nucleic acids ofdiffering sequence from which to select a desired ligand. The source ofa candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. Modified nucleotides, such as nucleotides withphotoreactive groups or other modifications, can be incorporated intothe candidate mixture. In addition, a SELEX process can be used toproduce a candidate mixture, that is, a first SELEX process experimentcan be used to produce a ligand-enriched mixture of nucleic acids thatis used as the candidate mixture in a second SELEX process experiment. Acandidate mixture can also comprise nucleic acids with one or morecommon structural motifs. As used herein, a candidate mixture is alsosometimes referred to as a “pool” or a “library.” For example, an “RNApool” refers to a candidate mixture comprised of RNA.

In various embodiments, each nucleic acid in a candidate mixture mayhave fixed sequences on either side of a randomized region, tofacilitate the amplification process. The nucleic acids in the candidatemixture of nucleic acids can each further comprise fixed regions or“tail” sequences at their 5′ and 3′ termini to prevent the formation ofhigh molecular weight parasites during the amplification process.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides of anylength, and such nucleotides may include deoxyribonucleotides,ribonucleotides, and/or analogs or chemically modifieddeoxyribonucleotides or ribonucleotides. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules.

If present, chemical modifications of a nucleotide can include, singlyor in any combination, 2′-position sugar modifications, 5-positionpyrimidine modifications (e.g.,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo- or 5-iodo-uracil, backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine, and the like.

In one embodiment, the term “C-5 modified pyrimidine” refers to apyrimidine with a modification at the C-5 position including, but notlimited to those moieties illustrated in FIG. 14. Examples of a C-5modified pyrimidine include those described in U.S. Pat. Nos. 5,719,273and 5,945,527. Examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent selected from:benzylcarboxyamide (alternatively benzylaminocarbonyl) (Bn),naphthylmethylcarboxyamide (alternatively naphthylmethylaminocarbonyl)(Nap), tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp),and isobutylcarboxyamide (alternatively isobutylaminocarbonyl) (iBu) asillustrated immediately below.

As delineated above, representative C-5 modified pyrimidines include:5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

Modifications can also include 3′ and 5′ modifications, such as cappingor pegylation. Other modifications can include substitution of one ormore of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and those with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylators, and those with modified linkages (e.g., alphaanomeric nucleic acids, etc.). Further, any of the hydroxyl groupsordinarily present in a sugar may be replaced by a phosphonate group ora phosphate group; protected by standard protecting groups; or activatedto prepare additional linkages to additional nucleotides or to a solidsupport. The 5′ and 3′ terminal OH groups can be phosphorylated orsubstituted with amines, organic capping group moieties of from about 1to about 20 carbon atoms, or organic capping group moieties of fromabout 1 to about 20 polyethylene glycol (PEG) polymers or otherhydrophilic or hydrophobic biological or synthetic polymers. If present,a modification to the nucleotide structure may be imparted before orafter assembly of a polymer. A sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

In one embodiment, the variable region of the aptamer includesnucleotides that include modified bases. Certain modified aptamers maybe used in any of the described methods, devices, and kits. Thesemodified nucleotides have been shown to produce novel aptamers that havevery slow off-rates from their respective targets while maintaining highaffinity to the target. In one embodiment, the C-5 position of thepyrimidine bases may be modified. Aptamers containing nucleotides withmodified bases have a number of properties that are different than theproperties of standard aptamers that include only naturally occurringnucleotides (i.e., unmodified nucleotides). In one embodiment, themethod for modification of the nucleotides includes the use of an amidelinkage. However, other suitable methods for modification may be used.It has been surprisingly observed that the structure of the identifiedslow off-rate aptamers does not appear to be entirely in accordance withthe structure predicted by standard base pairing models. Thisobservation is supported by the fact that the measured meltingtemperatures of the slow off-rate aptamers are not consistent with themelting temperatures predicted by the models, see FIG. 13. As shown,there appears to be no correlation between the measured and predictedmelting temperatures of the slow off-rate aptamers. On average, thecalculated melting temperature (Tm) is 6° C. lower than the measured Tm.The measured melting temperatures indicate that slow off-rate aptamersincluding these modified nucleotides are more stable than may bepredicted and potentially possess novel secondary structures. Thesemodified aptamers also have different circular dichorism spectra thancorresponding aptamers that include only unmodified nucleotides. In thecase of many targets, slow off-rate aptamers to the target are morelikely to be identified when modified nucleotides are used in theproduction of the initial library or candidate mixture.

As used herein, “modified nucleic acid” refers to a nucleic acidsequence containing one or more modified nucleotides. In someembodiments it may be desirable that the modified nucleotides arecompatible with the SELEX process.

“Polypeptide,” “peptide,” and “protein” are used interchangeably hereinto refer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and/or it maybe interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Polypeptides can besingle chains or associated chains.

As used herein, “photoreactive nucleotide” means any modified nucleotidethat is capable of photocrosslinking with a target, such as a protein,upon irradiation with certain wavelengths of light. For example,photoaptamers produced by the photoSELEX process can include aphotoreactive group selected from the following: 5-bromouracil (BrU),5-iodouracil (IU), 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil,4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine. A “photoreactive pyrimidine” means anymodified pyrimidine that is capable of photocrosslinking with a targetupon irradiation of certain wavelengths. Exemplary photoreactivepyrimidines include 5-bromo-uracil (BrdU), 5-bromo-cytosine (BrdC),5-iodo-uracil (IdU), and 5-iodo-cytosine (IdC). In various embodiments,the photoreactive functional group will absorb wavelengths of light thatare not absorbed by the target or the non-modified portions of theoligonucleotide.

“SELEX” refers to a process that combines the selection of nucleic acidsthat interact with a target in a desirable manner (e.g., binding to aprotein) with the amplification of those selected nucleic acids.Optional iterative cycling of the selection/amplification steps allowsselection of one or a small number of nucleic acids that interact moststrongly with the target from a pool that contains a very large numberof nucleic acids. Cycling of the selection/amplification procedure iscontinued until a selected goal is achieved. The SELEX methodology isdescribed in the SELEX Patents. In some embodiments of the SELEXprocess, aptamers that bind non-covalently to their targets aregenerated. In other embodiments of the SELEX process, aptamers that bindcovalently to their targets are generated.

As used herein the term “amplification” or “amplifying” means anyprocess or combination of process steps that increases the amount ornumber of copies of a molecule or class of molecules.

“SELEX target” or “target molecule” or “target” refers herein to anycompound upon which a nucleic acid can act in a desirable manner. ASELEX target molecule can be a protein, peptide, nucleic acid,carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, any portion or fragment of any ofthe foregoing, etc., without limitation. In one embodiment, a SELEXtarget does not include molecules that are known to bind nucleic acids,such as, for example, known nucleic acid binding proteins (e.g.transcription factors). Virtually any chemical or biological effectormay be a suitable SELEX target. Molecules of any size can serve as SELEXtargets. A target can also be modified in certain ways to enhance thelikelihood or strength of an interaction between the target and thenucleic acid. A target can also include any minor variation of aparticular compound or molecule, such as, in the case of a protein, forexample, minor variations in amino acid sequence, disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation or modification, such as conjugation with alabeling component, which does not substantially alter the identity ofthe molecule. A “target molecule” or “target” is a set of copies of onetype or species of molecule or multimolecular structure that is capableof binding to an aptamer. “Target molecules” or “targets” refer to morethan one such set of molecules. Embodiments of the SELEX process inwhich the target is a peptide are described in U.S. Pat. No. 6,376,190,entitled “Modified SELEX Processes Without Purified Protein,”incorporated herein by reference in its entirety. FIG. 7 lists over 500targets for which aptamers have been produced including a variety ofslow off-rate aptamers.

As used herein, “competitor molecule” and “competitor” are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule. In this context, non-targetmolecules include free aptamers, where, for example, a competitor can beused to inhibit the aptamer from binding (re-binding), non-specifically,to another non-target molecule. A “competitor molecule” or “competitor”is a set of copies of one type or species of molecule. “Competitormolecules” or “competitors” refer to more than one such set ofmolecules. Competitor molecules include, but are not limited tooligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmonsperm DNA, tRNA, dextran sulfate, polydextran, abasic phosphodiesterpolymers, dNTPs, and pyrophosphate). In various embodiments, acombination of one or more competitor can be used.

As used herein, “non-specific complex” refers to a non-covalentassociation between two or more molecules other than an aptamer and itstarget molecule. A non-specific complex represents an interactionbetween classes of molecules. Non-specific complexes include complexesformed between an aptamer and a non-target molecule, a competitor and anon-target molecule, a competitor and a target molecule, and a targetmolecule and a non-target molecule.

As used herein, the term “slow off-rate enrichment process” refers to aprocess of altering the relative concentrations of certain components ofa candidate mixture such that the relative concentration of aptameraffinity complexes having slow dissociation rates is increased relativeto the concentration of aptamer affinity complexes having faster, lessdesirable dissociation rates. In one embodiment, the slow off-rateenrichment process is a solution-based slow off-rate enrichment process.In this embodiment, a solution-based slow off-rate enrichment processtakes place in solution, such that neither the target nor the nucleicacids forming the aptamer affinity complexes in the mixture areimmobilized on a solid support during the slow off-rate enrichmentprocess. In various embodiments, the slow off-rate enrichment processcan include one or more steps, including the addition of and incubationwith a competitor molecule, dilution of the mixture, or a combination ofthese (e.g., dilution of the mixture in the presence of a competitormolecule). Because the effect of an slow off-rate enrichment processgenerally depends upon the differing dissociation rates of differentaptamer affinity complexes (i.e., aptamer affinity complexes formedbetween the target molecule and different nucleic acids in the candidatemixture), the duration of the slow off-rate enrichment process isselected so as to retain a high proportion of aptamer affinity complexeshaving slow dissociation rates while substantially reducing the numberof aptamer affinity complexes having fast dissociation rates. The slowoff-rate enrichment process may be used in one or more cycles during theSELEX process. When dilution and the addition of a competitor are usedin combination, they may be performed simultaneously or sequentially, inany order. The slow off-rate enrichment process can be used when thetotal target (protein) concentration in the mixture is low. In oneembodiment, when the slow off-rate enrichment process includes dilution,the mixture can be diluted as much as is practical, keeping in mind thatthe nucleic acids are recovered for subsequent rounds in the SELEXprocess. In one embodiment, the slow off-rate enrichment processincludes the use of a competitor as well as dilution, permitting themixture to be diluted less than might be necessary without the use of acompetitor.

In one embodiment, the slow off-rate enrichment process includes theaddition of a competitor, and the competitor is a polyanion (e.g.,heparin or dextran sulfate (dextran)). Heparin or dextran have been usedin the identification of specific aptamers in prior SELEX selections. Insuch methods, however, heparin or dextran is present during theequilibration step in which the target and aptamer bind to formcomplexes. In such methods, as the concentration of heparin or dextranincreases, the ratio of high affinity target/aptamer complexes to lowaffinity target/aptamer complexes increases. However, a highconcentration of heparin or dextran can reduce the number of highaffinity target/aptamer complexes at equilibrium due to competition fortarget binding between the nucleic acid and the competitor. By contrast,the presently described methods add the competitor after thetarget/aptamer complexes have been allowed to form and therefore doesnot affect the number of complexes formed. Addition of competitor afterequilibrium binding has occurred between target and aptamer creates anon-equilibrium state that evolves in time to a new equilibrium withfewer target/aptamer complexes. Trapping target/aptamer complexes beforethe new equilibrium has been reached enriches the sample for slowoff-rate aptamers since fast off-rate complexes will dissociate first.

In another embodiment, a polyanionic competitor (e.g., dextran sulfateor another polyanionic material) is used in the slow off-rate enrichmentprocess to facilitate the identification of an aptamer that isrefractory to the presence of the polyanion. In this context,“polyanionic refractory aptamer” is an aptamer that is capable offorming an aptamer/target complex that is less likely to dissociate inthe solution that also contains the polyanionic refractory material thanan aptamer/target complex that includes a non-polyanionic refractoryaptamer. In this manner, polyanionic refractory aptamers can be used inthe performance of analytical methods to detect the presence or amountor concentration of a target in a sample, where the detection methodincludes the use of the polyanionic material (e.g. dextran sulfate) towhich the aptamer is refractory.

Thus, in one embodiment, a method for producing a polyanionic refractoryaptamer is provided. In this embodiment, after contacting a candidatemixture of nucleic acids with the target, the target and the nucleicacids in the candidate mixture are allowed to come to equilibrium. Apolyanionic competitor is introduced and allowed to incubate in thesolution for a period of time sufficient to insure that most of the fastoff-rate aptamers in the candidate mixture dissociate from the targetmolecule. Also, aptamers in the candidate mixture that may dissociate inthe presence of the polyanionic competitor will be released from thetarget molecule. The mixture is partitioned to isolate the highaffinity, slow off-rate aptamers that have remained in association withthe target molecule and to remove any uncomplexed materials from thesolution. The aptamer can then be released from the target molecule andisolated. The isolated aptamer can also be amplified and additionalrounds of selection applied to increase the overall performance of theselected aptamers. This process may also be used with a minimalincubation time if the selection of slow off-rate aptamers is not neededfor a specific application.

Thus, in one embodiment a modified SELEX process is provided for theidentification or production of aptamers having slow (long) off-rateswherein the target molecule and candidate mixture are contacted andincubated together for a period of time sufficient for equilibriumbinding between the target molecule and nucleic acids contained in thecandidate mixture to occur. Following equilibrium binding an excess ofcompetitor molecule, e.g., polyanion competitor, is added to the mixtureand the mixture is incubated together with the excess of competitormolecule for a predetermined period of time. A significant proportion ofaptamers having off rates that are less than this predeterminedincubation period will dissociate from the target during thepredetermined incubation period. Re-association of these “fast” off-rateaptamers with the target is minimized because of the excess ofcompetitor molecule which can non-specifically bind to the target andoccupy aptamer binding sites on the target. A significant proportion ofaptamers having longer off rates will remain complexed to the targetduring the predetermined incubation period. At the end of the incubationperiod, partitioning nucleic acid-target complexes from the remainder ofthe mixture allows for the separation of a population of slow off-rateaptamers from those having fast off rates. A dissociation step can beused to dissociate the slow off-rate aptamers from their target andallows for isolation, identification, sequencing, synthesis andamplification of slow off-rate aptamers (either of individual aptamersor of a group of slow off-rate aptamers) that have high affinity andspecificity for the target molecule. As with conventional SELEX theaptamer sequences identified from one round of the modified SELEXprocess can be used in the synthesis of a new candidate mixture suchthat the steps of contacting, equilibrium binding, addition ofcompetitor molecule, incubation with competitor molecule andpartitioning of slow off-rate aptamers can be iterated/repeated as manytimes as desired.

The combination of allowing equilibrium binding of the candidate mixturewith the target prior to addition of competitor, followed by theaddition of an excess of competitor and incubation with the competitorfor a predetermined period of time allows for the selection of apopulation of aptamers having off rates that are much greater than thosepreviously achieved.

In order to achieve equilibrium binding, the candidate mixture may beincubated with the target for at least about 5 minutes, or at leastabout 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours or about 6hours.

The predetermined incubation period of competitor molecule with themixture of the candidate mixture and target molecule may be selected asdesired, taking account of factors such as the nature of the target andknown off rates (if any) of known aptamers for the target. Predeterminedincubation periods may be chosen from: at least about 5 minutes, atleast about 10 minutes, at least about 20 minutes, at least about 30minutes, at least 45 about minutes, at least about 1 hour, at leastabout 2 hours, at least about 3 hours, at least about 4 hours, at leastabout 5 hours, at least about 6 hours.

In other embodiments a dilution is used as an off rate enhancementprocess and incubation of the diluted candidate mixture, targetmolecule/aptamer complex may be undertaken for a predetermined period oftime, which may be chosen from: at least about 5 minutes, at least about10 minutes, at least about 20 minutes, at least about 30 minutes, atleast about 45 minutes, at least about 1 hour, at least about 2 hours,at least about 3 hours, at least about 4 hours, at least about 5 hours,at least about 6 hours.

Embodiments of the present disclosure are concerned with theidentification, production, synthesis and use of slow off-rate aptamers.These are aptamers which have a rate of dissociation (t_(1/2)) from anon-covalent aptamer-target complex that is higher than that of aptamersnormally obtained by conventional SELEX. For a mixture containingnon-covalent complexes of aptamer and target, the t_(1/2) represents thetime taken for half of the aptamers to dissociate from theaptamer-target complexes. The t_(1/2) of slow dissociation rate aptamersaccording to the present disclosure is chosen from one of: greater thanor equal to about 30 minutes; between about 30 minutes and about 240minutes; between about 30 minutes to about 60 minutes; between about 60minutes to about 90 minutes, between about 90 minutes to about 120minutes; between about 120 minutes to about 150 minutes; between about150 minutes to about 180 minutes; between about 180 minutes to about 210minutes; between about 210 minutes to about 240 minutes.

A characterizing feature of an aptamer identified by a SELEX procedureis its high affinity for its target. An aptamer will have a dissociationconstant (k_(d)) for its target that is chosen from one of: less thanabout 1 μM, less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 10 pM, less thanabout 1 pM.

“Tissue target” or “tissue” refers herein to a certain subset of theSELEX targets described above. According to this definition, tissues aremacromolecules in a heterogeneous environment. As used herein, tissuerefers to a single cell type, a collection of cell types, an aggregateof cells, or an aggregate of macromolecules. This differs from simplerSELEX targets that are typically isolated soluble molecules, such asproteins. In some embodiments, tissues are insoluble macromolecules thatare orders of magnitude larger than simpler SELEX targets. Tissues arecomplex targets made up of numerous macromolecules, each macromoleculehaving numerous potential epitopes. The different macromolecules whichcomprise the numerous epitopes can be proteins, lipids, carbohydrates,etc., or combinations thereof. Tissues are generally a physical array ofmacromolecules that can be either fluid or rigid, both in terms ofstructure and composition. Extracellular matrix is an example of a morerigid tissue, both structurally and compositionally, while a membranebilayer is more fluid in structure and composition. Tissues aregenerally not soluble and remain in solid phase, and thus partitioningcan be accomplished relatively easily. Tissue includes, but is notlimited to, an aggregate of cells usually of a particular kind togetherwith their intercellular substance that form one of the structuralmaterials commonly used to denote the general cellular fabric of a givenorgan, e.g., kidney tissue, brain tissue. The four general classes oftissues are epithelial tissue, connective tissue, nerve tissue andmuscle tissue.

Examples of tissues which fall within this definition include, but arenot limited to, heterogeneous aggregates of macromolecules such asfibrin clots which are a cellular; homogeneous or heterogeneousaggregates of cells; higher ordered structures containing cells whichhave a specific function, such as organs, tumors, lymph nodes, arteries,etc, and individual cells. Tissues or cells can be in their naturalenvironment, isolated, or in tissue culture. The tissue can be intact ormodified. The modification can include numerous changes such astransformation, transfection, activation, and substructure isolation,e.g., cell membranes, cell nuclei, cell organelles, etc.

Sources of the tissue, cell or subcellular structures can be obtainedfrom prokaryotes as well as eukaryotes. This includes human, animal,plant, bacterial, fungal, and viral structures.

As used herein, the term “labeling agent,” “label,” or “detectablemoiety”, or “detectable element” or “detectable component” refers to oneor more reagents that can be used to detect a target molecule/aptamercomplex. A detectable moiety or label is capable of being detecteddirectly or indirectly. In general, any reporter molecule that isdetectable can be a label. Labels include, for example, (i) reportermolecules that can be detected directly by virtue of generating asignal, (ii) specific binding pair members that may be detectedindirectly by subsequent binding to a cognate that contains a reportermolecule, (iii) mass tags detectable by mass spectrometry, (iv)oligonucleotide primers that can provide a template for amplification orligation, and (v) a specific polynucleotide sequence or recognitionsequence that can act as a ligand, such as, for example, a repressorprotein, wherein in the latter two instances the oligonucleotide primeror repressor protein will have, or be capable of having, a reportermolecule, and so forth. The reporter molecule can be a catalyst, such asan enzyme, a polynucleotide coding for a catalyst, promoter, dye,fluorescent molecule, quantum dot, chemiluminescent molecule, coenzyme,enzyme substrate, radioactive group, a small organic molecule,amplifiable polynucleotide sequence, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectable group, amass tag that alters the weight of the molecule to which it isconjugated for mass spectrometry purposes, and the like. The label canbe selected from electromagnetic or electrochemical materials. In oneembodiment, the detectable label is a fluorescent dye. Other labels andlabeling schemes will be evident to one skilled in the art based on thedisclosure herein.

A detectable moiety (element or component) can include any of thereporter molecules listed above and any other chemical or component thatmay be used in any manner to generate a detectable signal. Thedetectable moiety may be detected via a fluorescent signal, achemiluminescent signal, or any other detectable signal that isdependent upon the identity of the moiety. In the case where thedetectable moiety is an enzyme (for example, alkaline phosphatase), thesignal may be generated in the presence of the enzyme substrate and anyadditional factors necessary for enzyme activity. In the case where thedetectable moiety is an enzyme substrate, the signal may be generated inthe presence of the enzyme and any additional factors necessary forenzyme activity. Suitable reagent configurations for attaching thedetectable moiety to a target molecule include covalent attachment ofthe detectable moiety to the target molecule, non-covalent associationof the detectable moiety with another labeling agent component that iscovalently attached to the target molecule, and covalent attachment ofthe detectable moiety to a labeling agent component that isnon-covalently associated with the target molecule. Universal proteinstains (UPS) are described in detail in U.S. patent application Ser. No.10/504,696, filed Aug. 12, 2004, entitled “Methods and Reagents forDetecting Target Binding by Nucleic Acid Ligands”.

“Solid support” refers herein to any substrate having a surface to whichmolecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The substrate materials may be naturallyoccurring, synthetic, or a modification of a naturally occurringmaterial. Solid support materials may include silicon, graphite,mirrored surfaces, laminates, ceramics, plastics (including polymerssuch as, e.g., poly(vinyl chloride), cyclo-olefin copolymers,polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon,poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, etc.,either used by themselves or in conjunction with other materials.Additional rigid materials may be considered, such as glass, whichincludes silica and further includes, for example, glass that isavailable as Bioglass. Other materials that may be employed includeporous materials, such as, for example, controlled pore glass beads. Anyother materials known in the art that are capable of having one or morefunctional groups, such as any of an amino, carboxyl, thiol, or hydroxylfunctional group, for example, incorporated on its surface, are alsocontemplated.

The solid support may take any of a variety of configurations rangingfrom simple to complex and can have any one of a number of shapes,including a strip, plate, disk, rod, particle, including bead, tube,well, and the like. The surface may be relatively planar (e.g., aslide), spherical (e.g., a bead), cylindrical (e.g., a column), orgrooved. Exemplary solid supports that may be used include microtitrewells, microscope slides, membranes, paramagnetic beads, charged paper,Langmuir-Blodgett films, silicon wafer chips, flow through chips, andmicrobeads.

As used herein, “partitioning” means any process whereby one or morecomponents of a mixture are separated from other components of themixture. For example, aptamers bound to target molecules can bepartitioned from other nucleic acids that are not bound to targetmolecules and from non-target molecules. More broadly stated,partitioning allows for the separation of all the nucleic acids in acandidate mixture into at least two pools based on their relativeaffinity and/or dissociation rate to the target molecule. Partitioningcan be accomplished by various methods known in the art, includingfiltration, affinity chromatography, liquid-liquid partitioning, HPLC,etc. For example, nucleic acid-protein pairs can be bound tonitrocellulose filters while unbound nucleic acids are not. Columns thatspecifically retain nucleic acid-target complexes can also be used forpartitioning. For example, oligonucleotides able to associate with atarget molecule bound on a column allow the use of column chromatographyfor separating and isolating the highest affinity aptamers. Beads uponwhich target molecules are conjugated can also be used to partitionaptamers in a mixture. If the beads are paramagnetic, the partitioningcan be achieved through application of a magnetic field. Surface plasmonresonance technology can be used to partition nucleic acids in a mixtureby immobilizing a target on a sensor chip and flowing the mixture overthe chip, wherein those nucleic acids having affinity for the target canbe bound to the target, and the remaining nucleic acids can be washedaway. Liquid-liquid partitioning can be used as well as filtration gelretardation and density gradient centrifugation. Affinity tags on thetarget molecules can also be used to separate nucleic acid moleculesbound to the tagged target from aptamers that are free in solution. Forexample, biotinylated target molecules, along with aptamers bound tothem, can be sequestered from the solution of unbound nucleic acidsequences using streptavidin paramagnetic beads. Affinity tags can alsobe incorporated into the aptamer during preparation.

As used herein, “photoSELEX” is an acronym for Photochemical SystematicEvolution of Ligands by Exponential enrichment and refers to embodimentsof the SELEX process in which photocrosslinking aptamers are generated.In one embodiment of the photoSELEX process, a photoreactive nucleotideactivated by absorption of light is incorporated in place of a nativebase in either RNA- or in ssDNA-randomized oligonucleotide libraries,the nucleic acid target molecule mixture is irradiated causing somenucleic acids incorporated in nucleic acid-target molecule complexes tocrosslink to the target molecule via the photoreactive functionalgroups, and the selection step is a selection for photocrosslinkingactivity. The photoSELEX process is described in great detail in thePhotoSELEX Patents.

As used herein, “photoaptamer” and “photoreactive aptamer” are usedinterchangeably to refer to an aptamer that contains one or morephotoreactive functional groups that can covalently bind to or“crosslink” with a target molecule. For example, a naturally occurringnucleic acid residue may be modified to include a chemical functionalgroup that confers photoreactivity upon the nucleic acid residue uponexposure to a radiation source of an appropriate wavelength. In someembodiments, a photoreactive aptamer is identified initially. In otherembodiments, an aptamer is first identified and is subsequently modifiedto incorporate one or more photoreactive functional groups, therebygenerating a photoaptamer. In these embodiments, one or morephotoreactive nucleic acid residues can be incorporated into an aptamereither by substituting a photoreactive nucleic acid residue in the placeof one or more other nucleotides, such as one or more of the thymidineand/or cytidine nucleotides in the aptamer, for example, or by modifyingone or more nucleic acid residues to include a photoreactive functionalgroup.

Exemplary photoreactive functional groups that may be incorporated by aphotoaptamer include 5-bromouracil, 5-iodouracil, 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-thiouracil,4-thiocytosine, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine.

In addition to these exemplary nucleoside-based photoreactive functionalgroups, other photoreactive functional groups that can be added to aterminal end of an aptamer using an appropriate linker molecule can alsobe used. Such photoreactive functional groups include benzophenone,anthraquinone, 4-azido-2-nitro-aniline, psoralen, derivatives of any ofthese, and the like.

A photoreactive functional group incorporated by a photoaptamer may beactivated by any suitable method. In one embodiment, a photoaptamercontaining a photoreactive functional group can be crosslinked to itstarget by exposing the photoaptamer and its bound target molecule to asource of electromagnetic radiation. Suitable types of electromagneticradiation include ultraviolet light, visible light, X-rays, and gammarays. Suitable radiation sources include sources that utilize eithermonochromatic light or filtered polychromatic light.

As used herein, the term “the affinity SELEX process” refers toembodiments of the SELEX process in which non-photocrosslinking aptamersto targets are generated. In some embodiments of the affinity SELEXprocess, the target is immobilized on a solid support either before orafter the target is contacted with the candidate mixture of nucleicacids. The association of the target with the solid support allowsnucleic acids in the candidate mixture that have bound and in the casewhere a slow off-rate enrichment process is used, stay bound to thetarget to be partitioned from the remainder of the candidate mixture.The term “bead affinity SELEX process” refers to particular embodimentsof the affinity SELEX process where the target is immobilized on a bead,for example, before contact with the candidate mixture of nucleic acids.In some embodiments, the beads are paramagnetic beads. The term “filteraffinity SELEX process” refers to embodiments where nucleic acid targetcomplexes are partitioned from candidate mixture by virtue of theirassociation with a filter, such as a nitrocellulose filter. Thisincludes embodiments where the target and nucleic acids are initiallycontacted in solution, and contacted with the filter, and also includesembodiments where nucleic acids are contacted with target that ispre-immobilized on the filter. The term “plate affinity SELEX process”refers to embodiments where the target is immobilized on the surface ofa plate, such as, for example, a multi-well microtiter plate. In someembodiments, the plate is comprised of polystyrene. In some embodiments,the target is attached to the plate in the plate affinity SELEX processthrough hydrophobic interactions.

The present disclosure describes improved SELEX methods for generatingaptamers that are capable of binding to target molecules. Morespecifically, the present disclosure describes methods for identifyingaptamers and/or photoaptamers having slower rates of dissociation fromtheir respective targeted molecules than aptamers obtained with previousSELEX methods. The disclosure further describes aptamers and/orphotoaptamers obtained using the methods described herein and methods ofusing the same.

In one embodiment, a method is provided for identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising (a) preparing a candidate mixture of nucleic acid sequences;(b) contacting the candidate mixture with a target molecule whereinnucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; (c) applying a slow off-rate enrichmentprocess to allow the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; (d) partitioning theremaining nucleic acid-target molecule complexes from both free nucleicacids and non-target molecules in the candidate mixture; and (e)identifying an aptamer to the target molecule. The process may furtherinclude the iterative step of amplifying the nucleic acids that bind tothe target molecule to yield a mixture of nucleic acids enriched insequences that are able to bind to the target molecule yet producenucleic acid-target molecule complexes having slow dissociation rates.As defined above, the slow off-rate enrichment process can be selectedfrom (a) diluting the candidate mixture containing the nucleicacid-target molecule complexes; (b) adding at least one competitor tothe candidate mixture containing the nucleic acid-target moleculecomplexes, and diluting the candidate mixture containing the nucleicacid-target molecule complexes; (c) and adding at least one competitorto the candidate mixture containing the nucleic acid-target moleculecomplexes.

In one embodiment, a method is provided for producing an aptamer havinga slow rate of dissociation from its target molecule, the methodcomprising (a) preparing a candidate mixture of nucleic acid sequences;(b) contacting the candidate mixture with a target molecule whereinnucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; (c) applying a slow off-rate enrichmentprocess to allow the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; (d) partitioning theremaining nucleic acid-target molecule complexes from both free nucleicacids and non-target molecules in the candidate mixture; and (e)producing an aptamer to the target molecule. The process may furtherinclude the iterative step of amplifying the nucleic acids that bind tothe target molecule to yield a mixture of nucleic acids enriched insequences that are able to bind to the target molecule yet producenucleic acid-target molecule complexes having slow dissociation rates.As defined above, the slow off-rate enrichment process can be selectedfrom (a) diluting the candidate mixture containing the nucleicacid-target molecule complexes; (b) adding at least one competitor tothe candidate mixture containing the nucleic acid-target moleculecomplexes, and diluting the candidate mixture containing the nucleicacid-target molecule complexes; (c) and adding at least one competitorto the candidate mixture containing the nucleic acid-target moleculecomplexes.

In one embodiment, a method is provided for identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) incubating thecandidate mixture and target molecule together for a period of timesufficient to achieve equilibrium binding; (d) applying a slow off-rateenrichment process to allow the dissociation of nucleic acid-targetmolecule complexes with relatively fast dissociation rates to themixture of (c); (e) incubating the mixture of the candidate mixture, thenucleic acid-target molecule complexes and the competitor molecule from(d) for a predetermined period of time; (f) partitioning the nucleicacid-target molecule complexes from the candidate mixture; (g)dissociating the nucleic acid-target molecule complexes to generate freenucleic acids; (h) amplifying the free nucleic acids to yield a mixtureof nucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule may be identified. As defined above, theslow off-rate enrichment process can be selected from (a) diluting thecandidate mixture containing the nucleic acid-target molecule complexes;(b) adding at least one competitor to the candidate mixture containingthe nucleic acid-target molecule complexes, and diluting the candidatemixture containing the nucleic acid-target molecule complexes; (c) andadding at least one competitor to the candidate mixture containing thenucleic acid-target molecule complexes.

In another embodiment, a method is provided for producing an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) incubating thecandidate mixture and target molecule together for a period of timesufficient to achieve equilibrium binding; (d) applying a slow off-rateenrichment process to allow the dissociation of nucleic acid-targetmolecule complexes with relatively fast dissociation rates to themixture of (c); (e) incubating the mixture of the candidate mixture, thenucleic acid-target molecule complexes and the competitor molecule from(d) for a predetermined period of time; (f) partitioning the nucleicacid-target molecule complexes from the candidate mixture; (g)dissociating the nucleic acid-target molecule complexes to generate freenucleic acids; (h) amplifying the free nucleic acids to yield a mixtureof nucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule may be produced. As defined above, theslow off-rate enrichment process can be selected from (a) diluting thecandidate mixture containing the nucleic acid-target molecule complexes;(b) adding at least one competitor to the candidate mixture containingthe nucleic acid-target molecule complexes, and diluting the candidatemixture containing the nucleic acid-target molecule complexes; (c) andadding at least one competitor to the candidate mixture containing thenucleic acid-target molecule complexes.

In another embodiment, a method is provided of identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids, whereinthe candidate mixture comprises modified nucleic acids in which one,several or all pyrimidines in at least one, or each, nucleic acid of thecandidate mixture is chemically modified at the 5-position; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) partitioning theincreased affinity nucleic acids from the remainder of the candidatemixture; and (d) amplifying the increased affinity nucleic acids toyield a mixture of nucleic acids enriched in nucleic acid sequences thatare capable of binding to the target molecule with increased affinity,whereby an aptamer to the target molecule may be identified.

In another embodiment, a method is provided for producing an aptamerhaving a slow rate of dissociation from its target molecule, said methodcomprising preparing or synthesizing an aptamer that includes a nucleicacid sequence identified by the following process: (a) preparing acandidate mixture of nucleic acids, wherein the candidate mixturecomprises modified nucleic acids in which one, several or allpyrimidines in at least one, or each, nucleic acid of the candidatemixture is chemically modified at the 5-position; (b) contacting thecandidate mixture with a target molecule, wherein nucleic acids havingan increased affinity to the target molecule relative to other nucleicacids in the candidate mixture bind the target molecule, forming nucleicacid-target molecule complexes; (c) partitioning the increased affinitynucleic acids from the remainder of the candidate mixture; and (d)amplifying the increased affinity nucleic acids to yield a mixture ofnucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule is identified.

In another embodiment, a non-covalent complex of an aptamer and itstarget is provided, wherein the rate of dissociation (t_(1/2)) of theaptamer from the target is chosen from one of: greater than or equal toabout 30 minutes; between about 30 minutes and about 240 minutes; about30 minutes to about 60 minutes; about 60 minutes to about 90 minutes;about 90 minutes to about 120 minutes; about 120 minutes to about 150minutes; about 150 minutes to about 180 minutes; about 180 minutes toabout 210 minutes; about 210 minutes to about 240 minutes.

In another embodiment, a non-covalent complex of an aptamer and a targetis provided, wherein the aptamer has a K_(d) for the target of about 100nM or less, wherein the rate of dissociation (t_(1/2)) of the aptamerfrom the target is greater than or equal to about 30 minutes, andwherein one, several or all pyrimidines in the nucleic acid sequence ofthe aptamer are modified at the 5-position of the base. Themodifications may be selected from the group of compounds shown in FIG.14, these modifications are referred to as “base modified nucleotides”.Aptamers may be designed with any combination of the base modifiedpyrimidines desired.

Improved methods for performing SELEX with modified nucleotides,including nucleotides which contain photoactive groups or nucleotideswhich contain placeholders for photoactive groups are disclosed in U.S.application Ser. No. 12/175,388, entitled “Improved SELEX andPHOTOSELEX” which is being filed concurrently with the instantapplication and which is incorporated herein by reference in itsentirety. In another embodiment, the candidate mixture of nucleic acidmolecules includes nucleic acids containing modified nucleotide basesthat may aid in the formation of modified nucleic acid-target complexeswith relatively slow dissociation rates.

The various methods and steps described herein can be used to generatean aptamer capable of either (1) binding to a target molecule or (2)binding to a target molecule and subsequently forming a covalent linkagewith the target molecule upon irradiation.

Aptamers identified according to the methods described herein are usefulin a range of diagnostic and therapeutic methods. Slow off-rate aptamerswill bind to the target for a longer duration. This is useful indiagnostic methods where the binding of an aptamer to the target may beused to detect the presence, absence, amount or quantity of the targetmolecule and a prolonged interaction of the aptamer and targetfacilitates such detection. A similar advantage may be afforded whereslow off-rate aptamers are used in imaging methods, in vitro or in vivo.A prolonged interaction of aptamer and target also provides for improvedtherapeutic methods of treatment where the prolonged interaction mayallow for an improved therapeutic effect, e.g. owing to the longeractivation or inhibition of the target molecule or downstream signalingcascade.

Accordingly, in various embodiments, slow off-rate aptamers obtained,identified or produced by the described methods can be used in a varietyof methods of medical treatment or methods of diagnosis (in vitro or invivo). In one embodiment, slow off-rate aptamers can be used in a methodof treatment of disease. In one embodiment, slow off-rate aptamers canbe used in a method for diagnosis of disease in vivo. In anotherembodiment, slow off-rate aptamers can be used in vitro for thediagnosis of disease. In another embodiment, a slow off-rate aptamer canbe used in the manufacture of a therapeutic (e.g. pharmaceuticalcomposition) or the manufacture of a diagnostic agent for use in amethod of treatment or diagnosis of disease. Diagnostic or therapeuticapplications of slow off-rate aptamers may involve a diagnostic ortherapeutic outcome that depends on the specific and/or high affinitybinding of the slow off-rate aptamer to its target. Slow off-rateaptamers may also be used in target validation and high throughputscreening assays in the drug development process.

In one embodiment, slow off-rate aptamers are suitable reagents formolecular imaging in vivo. In this embodiment, a slow off-rate aptamermay be used in vivo to detect the presence of a pathology, diseaseprocess, or other condition in the body of an individual (e.g., a humanor an animal), where the binding of the aptamer to its target indicatesthe presence of the disease process or other condition. For example, anaptamer to the VEGF receptor may be used in vivo to detect the presenceof cancer in a particular area (e.g., a tissue, an organ, etc.) of thebody of an individual, as the VEGF receptor is abundantly expressedwithin tumors and their neovasculature, or an aptamer to the EGFreceptor may be used in vivo to detect the presence of cancer in aparticular area (e.g., a tissue, an organ, etc.) of the body of anindividual, as the EGF receptor is often expressed at high levels ontumor cells. That is, the molecular target will be the extracellulardomain (ECD) of an induced receptor, as such targets are located outsideof the cells and are accessible through the vasculature. Additionally,the ECDs tend to be localized at the site of pathology, even though somesmall fraction of the specific ECD may be shed through biologicalprocesses, including cell death.

The obvious candidates for molecular imaging, high affinity monoclonalantibodies, have not become the reagent of choice for this application.Molecular imaging reagents have precise requirements. They must havehigh binding activity for their intended target, and low bindingactivity for other targets in a human or animal. Slow off-rate aptamershave unique advantages that render them desirable for use in molecularimaging in vivo. On the one hand, they are selected to have slowdissociation rate constants, thus allowing residence in vivo on theintended target for a substantial length of time (at least about 30minutes). On the other hand, slow off-rate aptamers are expected to havevery fast clearance from the vasculature. Slow dissociation rateconstants and fast clearance from the vasculature are two desiredproperties for molecular imaging in vivo. From a kinetic prospective,good in vivo molecular imaging reagents must stay localized at the siteof the pathology while the free reagent concentration in the surroundingvasculature becomes low. This is a signal-to-noise constraint. Suitablesignal-to-noise ratios may be obtained by accumulation of signal at thesite of pathology in excess of the signal in the vasculature, or may beobtained by retention of a signal at the site of the pathology while thevasculature concentration is diminished.

Aptamers that do not have slow off-rate properties, of about the samemolecular weight and net charge as slow off-rate aptamers, have beenstudied in animals and humans for more than a decade. Generally, it hasbeen found that these aptamers clear from the vasculature quickly,usually by entering the kidney and/or the liver and then being furthermetabolized for excretion. Such aptamers show so-called “first pass”clearance unless high molecular weight adducts (such as, for example,PEG) are linked to the aptamers. Experiments have been done with anaptamer whose target is tenascin C, an extracellular protein (not anECD) found at high concentrations in some tumors. In those experiments,the tenascin C-specific aptamer cleared quickly and was able to beretained at the site of the tumor because the extracellular localconcentration of tenascin C is very high. Slow off-rate aptamers, bycontrast, will maintain the fast clearance rate of aptamers, but offer akinetic advantage due to their slow dissociation rates, rendering themsuitable for use with targets whose presence at the site of interest(e.g., the site of pathology) may be somewhat sparse (ECDs on tumors,for example).

Alternative reagents for molecular imaging do not share the two slowoff-rate aptamer properties (i.e., slow dissociation rate and fastclearance from the body). Monoclonal antibodies often have high affinityand specificity, and may have slow dissociation rate constants; however,monoclonal antibodies have very slow clearance rates from thevasculature. Short peptides, identified through, for example, phagedisplay, may have fast clearance but poor affinity and specificity andfast dissociation rates from their intended targets. Affibodies, aparticular peptide version of an antibody mimetic, may have reasonableaffinity and specificity and may have faster clearance than monoclonalantibodies, yet in order to achieve slow dissociation rates from theirtargets, affibodies are often made into dimers and higher ordermultimers, slowing their clearance at the same time that theirdissociation rates are enhanced.

Slow off-rate aptamers may be used for molecular imaging in vivo withone or more low molecular weight adducts to both protect the slowoff-rate aptamer from nucleases in the body and detect the intendedtarget once bound by the slow off-rate aptamer. For example, slowoff-rate aptamers may be attacked by nucleases in the blood, typicallyexonucleases (for DNA) that are easily blocked by using exonucleaserefractive adducts at the 5′ and 3′ terminal positions of the slowoff-rate aptamer, or endonucleases (for RNA) that are easily blocked byincorporating endonuclease refractive pyrimidines (such as, for example,2′ fluoro nucleotides) in the slow off-rate aptamer. Detection of theslow off-rate aptamer-target complex may be achieved by attaching adetection moiety to the slow off-rate aptamer. In some embodiments, thedetection moiety for these purposes may include cages for radioactivemolecules (e.g., technetium 99), clusters of iron for magnetic resonancedetection, isotopes of fluorine for PET imaging, and the like. Themodifications made to the slow off-rate aptamer to protect the integrityof the slow off-rate aptamer in the body and enable detection of theintended target should be designed such that they do not interfere withthe slow off-rate aptamer's interaction with its target and do not causethe slow off-rate aptamer to clear too slowly from the vasculature.

Diagnostic or assay devices, e.g. columns, test strips or biochips,having one or more slow off-rate aptamers adhered to a solid surface ofthe device are also provided. The aptamer(s) may be positioned so as tobe capable of binding target molecules that are contacted with the solidsurface to form aptamer-target complexes that remain adhered to thesurface of the device, thereby capturing the target and enablingdetection and optionally quantitation of the target. An array of slowoff-rate aptamers (which may be the same or different) may be providedon such a device.

In another embodiment, complexes including a slow off-rate aptamer and atarget molecule are provided. In other embodiments, a class of aptamerscharacterized by having high affinity for their corresponding targetmolecules and slow dissociation rates (t_(1/2)) from a non-covalentcomplex of the aptamer and target is provided.

With reference to FIG. 1A, the basic SELEX process generally begins withthe preparation of a candidate mixture of nucleic acids of differingsequence. The candidate mixture generally includes nucleic acidsequences that include two fixed regions (i.e., each of the members ofthe candidate mixture contains the same sequences in the same location)and a variable region. Typically, the fixed sequence regions areselected such that they assist in the amplification steps describedbelow, or enhance the potential of a given structural arrangement of thenucleic acids in the candidate mixture. The variable region typicallyprovides the target binding region of each nucleic acid in the candidatemixture, and this variable region can be completely randomized (i.e.,the probability of finding a base at any position being one in four) oronly partially randomized (e.g., the probability of finding a base atany location can be selected at any level between 0 and 100 percent).The prepared candidate mixture is contacted with the selected targetunder conditions that are favorable for binding to occur between thetarget and members of the candidate mixture. Under these conditions, theinteraction between the target and the nucleic acids of the candidatemixture generally forms nucleic acid-target pairs that have thestrongest relative affinity between members of the pair. The nucleicacids with the highest affinity for the target are partitioned fromthose nucleic acids with lesser affinity to the target. The partitioningprocess is conducted in a manner that retains the maximum number of highaffinity candidates. Those nucleic acids selected during partitioning ashaving a relatively high affinity to the target are amplified to createa new candidate mixture that is enriched in nucleic acids having arelatively high affinity for the target. By repeating the partitioningand amplifying steps above, the newly formed candidate mixture containsfewer and fewer unique sequences, and the average degree of affinity ofthe nucleic acid mixture to the target will generally increase. Taken toits extreme, the SELEX process will yield a candidate mixture containingone or a very small number of unique nucleic acids representing thosenucleic acids from the original candidate mixture that have the highestaffinity to the target molecule. However, this basic SELEX process doesnot select for aptamers that have slow off-rates from their targets.

The SELEX Patents and the PhotoSELEX Patents describe and elaborate onthis process in great detail. These patents include descriptions of thevarious targets that can be used in the process; methods for thepreparation of the initial candidate mixture; methods for partitioningnucleic acids within a candidate mixture; and methods for amplifyingpartitioned nucleic acids to generate enriched candidate mixtures. TheSELEX Patents also describe aptamer solutions obtained to a number ofdifferent types of target molecules, including protein targets whereinthe protein is and is not a nucleic acid binding protein.

With reference to FIG. 1B the modified SELEX process disclosed hereinincludes the introduction of a slow off-rate enrichment processfollowing equilibration of the candidate mixture of nucleic acids withthe target or targets and a partitioning step prior to subsequent stepsin the SELEX process. Introduction of a slow off-rate enrichment processto the basic SELEX process provides a means for enrichment of aptameraffinity complexes with slow dissociation rates from a set of nucleicacid-target complexes that includes a variety of dissociation rates.Thus, the modified SELEX process provides a method for identifyingaptamers that bind target molecules and, once bound, have relativelyslow rates of dissociation (also referred to herein as “off-rates”) fromthe target molecule.

As used herein “binding” generally refers to the formation of anon-covalent association between the ligand and the target, althoughsuch binding is not necessarily reversible. The terms “nucleicacid-target complex” or “complex” or “affinity complex” are used torefer to the product of such non-covalent binding association.

In various embodiments, the slow off-rate aptamers can be single- ordouble-stranded RNA or DNA oligonucleotides. The aptamers can containnon-standard or modified bases. Further, the aptamers can contain anytype of modification. As used herein, a “modified base” may include arelatively simple modification to a natural nucleic acid residue, whichmodification confers a change in the physical properties of the nucleicacid residue. Such modifications include, but are not limited to,modifications at the 5-position of pyrimidines, substitution withhydrophobic groups, e.g., benzyl, iso-butyl, indole, or naphthylmethyl,or substitution with hydrophilic groups, e.g., quaternary amine orguanidinium, or more “neutral” groups, e.g., imidazole and the like.Additional modifications may be present in the ribose ring, e.g.,2′-position, such as 2′-amino (2′-NH₂) and 2′-fluoro (2′-F), or thephosphodiester backbone, e.g., phosphorothioates or methyl phosphonates.

In various embodiments, a candidate mixture containing a randomized setof nucleic acid sequences containing modified nucleotide bases is mixedwith a quantity of the target molecule and allowed to establish bindingequilibrium with the target molecule. Generally, only some of thosenucleic acids that bind with high affinity to the target molecule willefficiently partition with the target.

In various embodiments, the candidate mixture includes nucleic acidsequences having variable regions that include modified groups. Themodified groups can be modified nucleotide bases. The variable regioncan contain fully or partially random sequences; it can also containsubportions of a fixed sequence that is incorporated within the variableregion. The nucleotides within the fixed regions can also containmodified nucleotide bases, or they can contain the standard set ofnaturally occurring bases.

In some embodiments, amplification occurs after members of the testmixture have been partitioned, and it is the nucleic acid that isamplified. For example, amplifying RNA molecules can be carried out by asequence of three reactions: making cDNA copies of selected RNAs, usingthe polymerase chain reaction to increase the copy number of each cDNA,and transcribing the cDNA copies to obtain RNA molecules having the samesequences as the selected RNAs. Any reaction or combination of reactionsknown in the art can be used as appropriate, including direct DNAreplication, direct RNA amplification and the like, as will berecognized by those skilled in the art. The amplification method mayresult in the proportions of the amplified mixture being representativeof the proportions of different sequences in the mixture prior toamplification. It is known that many modifications to nucleic acids arecompatible with enzymatic amplification. Modifications that are notcompatible with amplification can be made after each round ofamplification, if necessary.

The nucleic acid candidate mixture can be modified in various ways toenhance the probability of the nucleic acids having facilitatingproperties or other desirable properties, particularly those thatenhance the interaction between the nucleic acid and the target.Contemplated modifications include modifications that introduce otherchemical groups that have the correct charge, polarizability, hydrogenbonding, or electrostatic interaction to enhance the desiredligand-target interactions. The modifications that may enhance thebinding properties, including the affinity and/or dissociation rates, ofthe nucleic acid, for example, include hydrophilic moieties, hydrophobicmoieties, rigid structures, functional groups found in proteins such asimidazoles, primary alcohols, carboxylates, guanidinium groups, aminogroups, thiols and the like. Modifications can also be used to increasethe survival of aptamer-target complexes under stringent selectionpressures that can be applied to produce slow off-rate aptamers to awide range of targets. In one embodiment, BndU(5-(N-benzylcarboxyamide)-dU) is used in the generation of the candidatemixtures used to produce slow off-rate aptamers, although other modifiednucleotides are well suited to the production of such aptamers. Othermodified nucleotides are shown in FIG. 14.

A modified nucleotide candidate mixture for the purpose of thisapplication is any RNA or DNA candidate mixture that includes bothnaturally occurring and other than the naturally occurring nucleotides.Suitable modifications include modifications on every residue of thenucleic acid, on a single residue of the nucleic acid, on randomresidues, on all pyrimidines or all purines, on all occurrences of aspecific base (i.e., G, C, A, T or U) in the nucleic acid, or any othermodification scheme that may be suitable for a particular application.It is recognized that modification is not a prerequisite forfacilitating activity or binding ability of the aptamers. Aptamers mayinclude modified dUTP and dCTP residues.

Candidate mixtures for slow off-rate aptamers may comprise a set ofpyrimidines having a different modification at the C-5 base position.The C-5 modification may be introduced through an amide linkage,directly, or indirectly, or through another type of linkage. Thesecandidate mixtures are used in a SELEX process to identify slow off-rateaptamers. This process may be also include the use of the slow off-rateenrichment process. Candidate mixtures may be produced enzymatically orsynthetically.

As described above, the nucleotides can be modified in any number ofways, including modifications of the ribose and/or phosphate and/or basepositions. Certain modifications are described in U.S. Pat. No.5,660,985 entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides,” U.S. Pat. No. 5,428,149 entitled “Method forPalladium Catalyzed Carbon-Carbon Coupling and Products,” U.S. Pat. No.5,580,972 entitled “Purine Nucleoside Modifications by PalladiumCatalyzed Methods,” all of which are incorporated by reference herein.In one embodiment, modifications are those wherein another chemicalgroup is attached to the 5-position of a pyrimidine or the 2′ positionof a sugar. There is no limitation on the type of other chemical groupthat can be incorporated on the individual nucleotides. In someembodiments, the resulting modified nucleotide is amplifiable or can bemodified subsequent to the amplification steps (see, e.g., U.S. Pat. No.6,300,074 entitled “Systematic evolution of ligands by exponentialenrichment: Chemi-SELEX”).

In yet other embodiments, certain nucleotides are modified to produceaptamers that bind and form a covalent crosslink to their targetmolecule upon photo-activation of the affinity complex. This methodencompasses aptamers that bind, photocrosslink, and/or photoinactivatetarget molecules. In various embodiments, the aptamers containphotoreactive groups that are capable of photocrosslinking to the targetmolecule upon irradiation with light. In other embodiments, the aptamersare capable of bond formation with the target in the absence ofirradiation.

A photoreactive group can be any chemical structure that contains aphotochromophore and that is capable of photocrosslinking with a targetmolecule. Although referred to herein as a photoreactive group, in somecases, as described below, irradiation is not necessary for covalentbinding to occur between the aptamer and the target. In someembodiments, the photoreactive group will absorb light of a wavelengththat is not absorbed by the target or the non-modified portions of theoligonucleotide. Photoreactive groups include 5-halo-uridines,5-halo-cytosines, 7-halo-adenosines, 2-nitro-5-azidobenzoyls,diazirines, aryl azides, fluorinated aryl azides, benzophenones,amino-benzophenones, psoralens, anthraquinones, etc.

The photoreactive groups generally form bonds with the target uponirradiation of the associated nucleic acid-target pair. In some cases,irradiation is not required for bond formation to occur. Thephotocrosslink that typically occurs will be the formation of a covalentbond between the associated aptamer and the target. However, a tightionic interaction between the aptamer and target may also occur uponirradiation.

In one embodiment, photocrosslinking occurs due to exposure toelectromagnetic radiation. Electromagnetic radiation includesultraviolet light, visible light, X-ray, and gamma ray.

In various other embodiments, a limited selection of oligonucleotidesusing a SELEX method is followed by selection using a photoSELEX method.The initial SELEX selection rounds are conducted with oligonucleotidescontaining photoreactive groups. After a number of SELEX rounds,photoSELEX is conducted to select oligonucleotides capable of bindingthe target molecule.

In another embodiment, the production of an aptamer that includes acleavable or releasable section (also described as an element orcomponent) in the aptamer sequence is described. These additionalcomponents or elements are structural elements or components thatintroduce additional functionality into the aptamer and are thusfunctional elements or components. The aptamer is further produced withone or more of the following additional components (also described as afunctional or structural element or component or moiety in anycombination of these terms): a labeled or detectable component, a spacercomponent, and a specific binding tag or immobilization element orcomponent.

As noted above, the present disclosure provides methods for identifyingaptamers that bind target molecules and once bound have slow rates ofdissociation or off-rates. The slow off-rates obtained with this methodcan exceed a half-life of about one hour and as much as about 240minutes, that is, once a set of nucleic acid-target complexes isgenerated, half of the complexes in the set remain bound after one hour.Because the effect of a slow off-rate enrichment process depends uponthe differing dissociation rates of aptamer affinity complexes, theduration of the slow off-rate enrichment process is chosen so as toretain a high proportion of aptamer affinity complexes with slowdissociation rates while substantially reducing the number of aptameraffinity complexes with fast dissociation rates. For example, incubatingthe mixture for relatively longer periods of time after imposing theslow off-rate enrichment process will select for aptamers with longerdissociation rates than aptamers selected using slow off-rate enrichmentprocess having shorter incubation periods.

In various embodiments, the candidate mixture is mixed with a quantityof the target molecule and allowed to establish binding equilibrium withthe target molecule. Prior to partitioning the target bound nucleicacids from those free in solution, a slow off-rate enrichment process isimposed to enrich the bound population for slow dissociation rates. Asnoted above, the slow off-rate enrichment process can be applied by theaddition of a competitor molecule, by sample dilution, by a combinationof sample dilution in the presence of a competitor molecule. Thus, inone embodiment, the slow off-rate enrichment process is applied byintroducing competitor molecules into the mixture containing the nucleicacid-target complexes and incubating the mixture for some period of timebefore partitioning free from bound nucleic acids. The amount ofcompetitor molecules is generally at least one order of magnitude higherthan that of the nucleic acid molecules and may be two or more orders ofmagnitude higher. In another embodiment, the slow off-rate enrichmentprocess is applied by diluting the sample mixture of nucleic acid-targetcomplexes several fold (e.g. at least about one of 2×, 3×, 4×, 5×) involume and incubating the mixture for some period of time beforepartitioning free from bound nucleic acids. The dilution volume isgenerally at least one order of magnitude higher, and may be about twoor more orders of magnitude higher, than the original volume. In yetanother embodiment, a combination of both competitor molecules anddilution is used to apply the slow off-rate enrichment process. Inanother embodiment, candidate mixtures that have been shown to result inan increased frequency of slow dissociation aptamers are used to selecta number of candidate aptamers. These aptamers are screened to identifyslow dissociation rate aptamers.

In another embodiment, a slow off-rate aptamer that includes a cleavableor releasable section in the fixed region of the aptamer is produced.The aptamer can also be produced with one or more of the followingadditional components: a labeled component, a spacer component, and aspecific binding tag. Any or all of these elements may be introducedinto a single stranded aptamer. In one embodiment, the element isintroduced at the 5′ end of the aptamer. In another embodiment, one ormore of these elements is included by creating a partially doublestranded aptamer, where one strand contains the various elements desiredas well as a sequence complementary to one of the fixed sequencesections of the second strand containing the variable target bindingregion.

A “releasable” or “cleavable” element or moiety or component refers to afunctional group where certain bonds in the functional group can bebroken to produce 2 separate components. In various embodiments, thefunctional group can be cleaved by irradiating the functional group(photocleavable) at the appropriate wavelength or by treatment with theappropriate chemical or enzymatic reagents. In another embodiment, thereleasable element may be a disulfide bond that can be treated with areducing agent to disrupt the bond. The releasable element allows anaptamer/target affinity complex that is attached to a solid support tobe separated from the solid support, such as by elution of the complex.The releasable element may be stable to the conditions of the rest ofthe assay and may be releasable under conditions that will not disruptthe aptamer/target complex.

As disclosed herein, an aptamer can further comprise a “tag” or“immobilization component or element” or “specific binding component orelement” which refers to a component that provides a means for attachingor immobilizing an aptamer (and any target molecule that is bound to it)to a solid support. A “tag” is a set of copies of one type or species ofcomponent that is capable of associating with a probe. “Tags” refers tomore than one such set of components. The tag can be attached to orincluded in the aptamer by any suitable method. Generally, the tagallows the aptamer to associate, either directly or indirectly, with aprobe or receptor that is attached to the solid support. The probe maybe highly specific in its interaction with the tag and retain thatassociation during all subsequent processing steps or procedures. A tagcan enable the localization of an aptamer affinity complex (or optionalcovalent aptamer affinity complex) to a spatially defined address on asolid support. Different tags, therefore, can enable the localization ofdifferent aptamer covalent complexes to different spatially definedaddresses on a solid support. A tag can be a polynucleotide, apolypeptide, a peptide nucleic acid, a locked nucleic acid, anoligosaccharide, a polysaccharide, an antibody, an affybody, an antibodymimic, a cell receptor, a ligand, a lipid, biotin, any fragment orderivative of these structures, any combination of the foregoing, or anyother structure with which a probe (or linker molecule, as describedbelow) can be designed or configured to bind or otherwise associate withspecificity. Generally, a tag is configured such that it does notinteract intramolecularly with either itself or the aptamer to which itis attached or of which it is a part. If SELEX is used to identify anaptamer, the tag may be added to the aptamer either pre- or post-SELEX.The tag is included on the 5′-end of the aptamer post-SELEX, or the tagis included on the 3′-end of the aptamer post-SELEX, or the tags may beincluded on both the 3′ and 5′ ends of the aptamers in a post-SELEXprocess.

As illustrated in FIG. 8D, a fluorescent dye (such as Cy3), thephotocleavable and biotin moieties are all added to the end of theaptamer. Because of potential interactions between the photocleavablemoiety and the dye, a spacer is inserted between these two moieties. Allconstructs can be synthesized using standard phosphoramidite chemistry.Representative aptamer constructs are shown in FIG. 9A through FIG. 9F.The functionality can be split between the 5′ and 3′ end or combined oneither end. In addition to photocleavable moieties, other cleavablemoieties can be used, including chemically or enzymatically cleavablemoieties. A variety of spacer moieties can be used and one or morebiotin moieties can be included. Tags (also referred to asimmobilization or specific binding elements or components) other thanbiotin can also be incorporated. Suitable construction reagents includebiotin phosphoramidite, PC Linker (Glen Research PN 10-4920-02); PCbiotin phosphoramidite (Glen Research PN 10-4950-02); dSpacer CEphosphoramidite (Glen Research PN 10-1914-02); Cy3 phosphoramidite (GlenResearch PN 10-5913-02); and Arm26-Ach Spacer Amidite (Fidelity SystemsPN SP26Ach-05).

In one embodiment, base modifications of the nucleotides are used in theproduction of the variable region of the aptamer. These modifiednucleotides have been shown to produce aptamers that have very slowoff-rates from their targets.

In the methods of the present disclosure the candidate mixture maycomprise modified nucleic acids in which one, several (e.g. one of, orat least one of, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) or all pyrimidinesin at least one, or each, nucleic acid of the candidate mixture ischemically modified at the 5-position. Optionally, all C residues in thenucleic acids of the candidate mixture are chemically modified at the5-position. Optionally, all T residues in the nucleic acids of thecandidate mixture are chemically modified at the 5-position. Optionally,all U residues in the nucleic acids of the candidate mixture arechemically modified at the 5-position.

In another embodiment, the slow off-rate aptamers are mixed or exposedto a sample. The slow off-rate aptamer is allowed to react with, or bindto, its specific target in the sample to form a complex. A variety ofmethods may be used to detect either the target or the aptamer. Thetarget may be detected in the complex or upon liberation from thecomplex. The aptamer may be detected in the complex or upon liberationfrom the complex. The aptamer/target complex may be used to isolate thespecific target from other components in the test sample. Multipleaptamers may be used when a multiplexed assay for the detection of avariety of targets is desired.

The method of the instant disclosure is illustrated generally inExamples 1-8. Example 1 describes the general affinity SELEX methodusing a candidate mixture comprised of modified nucleotides. Example 2describes a photo SELEX method using a candidate mixture comprised ofmodified nucleotides and a 5′-terminal photoreactive group, and theimproved SELEX method in which dilution is used to provide the slowoff-rate enrichment process to the equilibrated aptamer:target mixture.Example 3 extends the method described in Example 2 by the addition of acompetitor to the dilution step. Example 4 illustrates the effectivenessof the slow off-rate enrichment process. The average dissociationhalf-life value (t_(1/2)) for aptamers using the modified nucleotides5-(N-benzylcarboxyamide)-dUTP (BndUTP), 5-(N-isobutylcarboxyamide)-dUTP(iBudUTP), or 5-(N-tryptaminocarboxyamide)-dUTP selected in the absenceof a slow off-rate enrichment process was 20 minutes with some aptamershaving a t_(1/2) value of up to one hour (FIG. 3A). This issubstantially longer than what has been previously described withnatural bases or other modified nucleotides. The average for aptamersselected with a slow off-rate enrichment process was over 85 minutes.More specifically, with reference to FIG. 3B, it can be seen thatintroduction of a slow off-rate enrichment process produced aptamerswith t_(1/2) values of about ≥30 min., ≥about 60 min., ≥about 90 min.,≥about 120 min., ≥about 150 min., ≥about 180 min., ≥about 210 min., and≥about 240 min. These dissociation rates for aptamer:target complexesare unprecedented.

Example 5 describes the generation of slow off-rate aptamers using aNapdU (5-(N-naphthylmethylcarboxyamide)-dUP) candidate mixture.

Example 6 describes the generation of a slow off-rate aptamer to apeptide target.

Example 7 illustrates the utility of slow off-rate aptamers relative toconventional aptamers.

Example 8 further illustrates the generation of slow off-rate aptamersusing a BndU candidate mixture.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention as defined in theappended claims.

Example 1. Incorporation of Modified Nucleotides in Nucleic AcidLibraries Leads to Higher Affinity Enriched Libraries in Affinity SELEX

A. Preparation of Candidate Mixtures

Candidate mixtures were prepared with dATP, dGTP, 5-methyl-dCTP (MedCTP)and either dTTP or one of three dUTP analogs:5-(N-benzylcarboxyamide)-dUTP (BndUTP), 5-(N-isobutylcarboxyamide)-dUTP(iBudUTP), or 5-(N-tryptaminocarboxyamide)-dUTP (TrpdUTP). Candidatemixtures were prepared by polymerase extension of a primer annealed to abiotinylated template (FIG. 2). For each candidate mixture composition,4.8 nmol forward PCR primer and 4 nmol template were combined in 100 μL1×KOD DNA Polymerase Buffer (Novagen), heated to 95° C. for 8 minutes,and cooled on ice. Each 100 μL primer:template mixture was added to a400 μL extension reaction containing 1×KOD DNA Polymerase Buffer, 0.125U/μL KOD XL DNA Polymerase, and 0.5 mM each dATP, MedCTP, dGTP, and dTTPor dUTP analog, and incubated at 70° C. for 30 minutes. Double-strandedproduct was captured via the template strand biotins by adding 1 mLstreptavidin-coated magnetic beads (MagnaBind Streptavidin, Pierce, 5mg/mL in 1M NaCl+0.05% TWEEN-20) and incubating at 25° C. for 10 minuteswith mixing. Beads were washed three times with 0.75 mL SB1T Buffer (40mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂), 0.05%TWEEN-20). The aptamer strand was eluted from the beads with 1.2 mL 20mM NaOH, neutralized with 0.3 mL 80 mM HCl, and buffered with 15 μL 1 MHEPES, pH 7.5. Candidate mixtures were concentrated with a Centricon-30to approximately 0.2 mL, and quantified by UV absorbance spectroscopy.

B. Immobilization of Target Proteins

Target proteins were purchased with poly His tags, such as, (His)₆ tags(R&D Systems) and immobilized on Co⁺²-NTA paramagnetic beads (MyOneTALON, Invitrogen, or hereinafter referred to as Talon beads). Targetproteins were diluted to 0.2 mg/mL in 0.5 mL B/W Buffer (50 mMNa-phosphate, pH 8.0, 300 mM NaCl, 0.01% TWEEN-20), and added to 0.5 mLTALON beads (pre-washed three times with B/W Buffer and resuspended to10 mg/mL in B/W Buffer). The mixture was rotated for 30 minutes at 25°C. and stored at 4° C. until use. TALON beads coated with (His)₆ peptidewere also prepared and stored as above. Prior to use, beads were washed3 times with B/W Buffer, once with SB1T, and resuspended in SB1T.

C. Aptamer Selection Scheme

Affinity selections were performed separately with each candidatemixture, comparing binding between target protein beads (signal, S) and(His)₆ beads (background, B). For each sample, a 0.5 μM candidate DNAmixture was prepared in 40 μL SB1T. 1 (His)₆-complement oligo (1 mM)(FIG. 2) was added to the DNA, along with 10 μL of a protein competitormixture (0.1% HSA, 10 μM casein, and 10 μM prothrombin in SB1T).

Binding reactions were performed by adding 50 μL target protein-coatedbeads or (His)₆-coated beads (5 mg/mL in SB1T) to the DNA mixture andincubating 37° C. for 15 minutes with mixing. The DNA solution wasremoved and the beads were washed 5 times at 37° C. with SB1T containing0.1 mg/mL herring sperm DNA (Sigma-Aldrich). Unless indicated, allwashes were performed by re-suspending the beads in 100 μL washsolution, mixing for 30 seconds, separating the beads with a magnet, andremoving the wash solution. Bound aptamers were eluted from the beads byadding 100 μL SB1T+2 M Guanidine-HCl and incubating at 37° C. for 5minutes with mixing. The aptamer eluate was transferred to a new tubeafter magnetic separation. After the first two selection rounds, thefinal two of five target beads washes were done for 5 minutes instead of30 seconds.

Primer beads were prepared by immobilizing biotinylated reverse PCRprimer to streptavidin-coated paramagnetic beads (MyOne-Streptavidin Cl(SA beads), Invitrogen). 5 mL SA beads (10 mg/mL) were washed once withNaClT (5 M NaCl, 0.01% TWEEN-20), and resuspended in 5 mL biotinylatedreverse PCR primer (5 μM in NaClT). The sample was incubated at 25° C.for 15 minutes, washed twice with 5 mL NaClT, resuspended in 12.5 mLNaClT (4 mg/mL), and stored at 4° C.

25 μL primer beads (4 mg/mL in NaClT) were added to the 100 μL aptamersolution in Guanidine Buffer and incubated at 50° C. for 15 minutes withmixing. The aptamer solution was removed, and the beads were washed 5times with SB1T. Aptamer was eluted from the beads by adding 85 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. 80 μL aptamereluate was transferred to a new tube after magnetic separation,neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5M Tris-HCl,pH 7.5.

D. Aptamer Amplification and Purification

Selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA wasadded to 12 μL QPCR Mix (5×KOD DNA Polymerase Buffer, 25 mM MgCl₂, 10 μMforward PCR primer, 10 μM biotinylated reverse PCR primer, 5×SYBR GreenI, 0.125 U/μL KOD XL DNA Polymerase, and 1 mM each dATP, dCTP, dGTP, anddTTP) and thermal cycled in an ABI5700 QPCR instrument with thefollowing protocol: 1 cycle of 99.9° C., 15 seconds, 55° C., 10 seconds,70° C., 30 minutes; 30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.Quantification was done with the instrument software and the number ofcopies of DNA selected with target beads and (His)₆ beads were comparedto determine signal/background ratios.

Following amplification, the PCR product was captured on SA beads viathe biotinylated antisense strand. 1.25 mL SA beads (10 mg/mL) werewashed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL SB1T, resuspendedin 2.5 mL 3 M NaCl, and stored at 4° C. 25 μL SA beads (4 mg/mL in 3 MNaCl) were added to 50 μL double-stranded QPCR product and incubated at25° C. for 5 minutes with mixing. The beads were washed once with SB1T,and the “sense” strand was eluted from the beads by adding 200 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. The elutedstrand was discarded and the beads were washed 3 times with SB and oncewith 16 mM NaCl.

Aptamer sense strand was prepared with the appropriate nucleotidecomposition by primer extension from the immobilized antisense strand.The beads were resuspended in 20 μL primer extension reaction mix (1×Primer Extension Buffer (120 mM Tris-HCl, pH 7.8 @ 20, 10 mM KCl, 7 mMMgSO₄, 6 mM (NH₄)₂SO₄, 0.001% BSA, and 0.01% Triton X100), 5 μM forwardPCR primer, 0.125 U/μL KOD XL DNA Polymerase, 0.5 mM each dATP, MedCTP,dGTP, and either dTTP or dUTP analog) and incubated at 68° C. for 30minutes with mixing. The beads were washed 3 times with SB1T, and theaptamer strand was eluted from the beads by adding 85 μL 20 mM NaOH, andincubating at 37° C. for 1 minute with mixing. 80 μL aptamer eluate wastransferred to a new tube after magnetic separation, neutralized with 20μL 80 mM HCl, and buffered with 5 μL 0.1 M HEPES, pH 7.5.

E. Selection Stringency and Feedback

The relative target protein concentration of the selection step waslowered each round in response to the SB ratio as follows, where signalS and background B are defined in Section C above:

if S/B<10,[P](i+1)=[P]i

if 10≤S/B<100,[NP](i+1)=[P]i/3.2

if S/B≥100,[NP](i+1)=[P]i/10

where [P]=protein concentration and i=current round number.

Target protein concentration was lowered by adjusting the mass of targetprotein beads (and (His)₆ beads for background determination) added tothe selection step.

After each selection round, the convergence state of the enriched DNAmixture was determined. 5 μL double-stranded QPCR product was diluted to200 μL with 4 mM MgCl₂ containing 1×SYBR Green I. Samples were overlaidwith 75 μL silicon oil and analyzed for convergence using a Cot analysiswhich measures the hybridization time for complex mixtures of doublestranded oligonucleotides. The sample was thermal cycled with thefollowing protocol: 3 cycles of 98° C., 1 minute, 85° C., 1 minute; 1cycle of 93° C., 1 minute, 85° C., 15 minutes. During the 15 minutes at85° C., fluorescent images were measured at 5-second intervals. Thefluorescence intensity was plotted as a function of log (time) toevaluate the diversity of the sequences.

F. Measurement of Equilibrium Binding Constant (Kd)

Equilibrium binding constants of the enriched libraries were measuredusing TALON bead partitioning. DNA was renatured by heating to 95° C.and slowly cooling to 37° C. Complexes were formed by mixing a lowconcentration of radiolabled DNA (˜1×10⁻¹¹ M) with a range ofconcentrations of target protein (1×10⁻⁷ M to 1×10⁻¹² M final) in SB1Buffer, and incubating at 37° C. A portion of each reaction wastransferred to a nylon membrane and dried to determine total counts ineach reaction. A small amount of 5 mg/mL TALON beads was added to theremainder of each reaction and mixed at 37° C. for one minute. A portionwas passed through a MultiScreen HV Plate (Millipore) under vacuum toseparate protein-bound complexes from unbound DNA and washed with 100 μLSB1 Buffer. The nylon membranes and MultiScreen HV Plates werephosphorimaged and the amount of radioactivity in each sample quantifiedusing a FUJI FLA-3000. The fraction of captured DNA was plotted as afunction of protein concentration and a non-linear curve-fittingalgorithm was used to extract equilibrium binding constants (K_(d)values) from the data. Table 1 shows the K_(d) values determined foreach enriched candidate mixture to a set of targets. NT indicates thatthe enriched library for a particular base composition did not appear tohave changed from the original candidate mixture, as determined by Cotanalysis, and was therefore Not Tested (NT).

Table 1 shows the equilibrium binding constants (K_(d)) for enrichedpools to fifteen different protein targets and four different DNAlibraries: naturally occurring bases (dT), 5-(N-benzylcarboxyamide)(BndU), 5-(N-isobutylcarboxyamide) (iBudU) or5-(N-tryptaminocarboxyamide) (TrpdU). An aptamer with a K_(d) of lessthan 1×10⁻⁸ is desirable. The use of modified bases in the SELEX processproduces a significantly higher percentage of desirable high affinityaptamers. It was observed that only 2 of the 14 aptamers produced withthe normal nucleotides have the desired slow dissociation rates. Slowoff-rate aptamers produced with the modified nucleotides were identified9 of 14, 7 of 14, and 14 of 14 for BndUTP, iBudUTP, and TrpdUTP,respectively.

TABLE 2 Equilibrium binding constants (K_(d)) of the enriched librariesselected with different chromophores, reported in units of molarity.Measurements were not made on libraries that failed to converge(indicated with an x). Target Protein BrdU AQ ANA Psor β-catenin 2.7. ×10⁻⁸ 3.6. × 10⁻⁹  1.1. × 10⁻⁹  1.6. × 10⁻⁹ bFGF 3.1. × 10⁻⁸ 5.7. × 10⁻¹⁰7.1. × 10⁻¹⁰  5.1. × 10⁻¹⁰ CMP-SAS x 6.2. × 10⁻⁹  7.3. × 10⁻⁹  4.9. ×10⁻⁸ endostatin 1.3. × 10⁻⁹ 8.7. × 10⁻¹⁰ 8.8. × 10⁻¹⁰ 1.3. × 10⁻⁹ IL-61.0. × 10⁻⁹ 5.4. × 10⁻¹⁰ 4.0. × 10⁻¹⁰ x myeloper-  6.0. × 10⁻¹⁰ 2.8. ×10⁻¹⁰ 5.0. × 10⁻¹⁰  1.5. × 10⁻¹⁰ oxidase SDF-1β  8.1. × 10⁻¹⁰ 5.7. ×10⁻¹⁰ 3.8. × 10⁻¹⁰ x TIMP-1 5.2. × 10⁻⁹ 7.3. × 10⁻⁹  8.9. × 10⁻⁹  x VEGF 7.2. × 10⁻¹⁰ 4.2. × 10⁻⁹  5.5. × 10⁻¹⁰ x vWF 2.6. × 10⁻⁸ 8.8. × 10⁻⁹ 8.1. × 10⁻⁹  x

Example 2. Generation of PhotoAptamers Using 5′-Fixed PhotoSELEX andSlow Off-Rate Enrichment Process by Dilution

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP were preparedby polymerase extension of a primer annealed to a biotinylated template(FIG. 4A-B). For each template, four different forward primers wereused, each possessing a unique chromophore at the 5′ terminus (see FIG.5 for the chromophore structures). For each candidate mixture, 11 nmolforward primer (with 5′ chromophore) and 10 nmol template were combinedin 250 μL Primer Extension Buffer (120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6mM (NH₄)₂SO₄, 7 mM MgSO₄, 0.1 mg/mL BSA, 0.1% Triton X-100), heated to95° C. for 5 minutes, and cooled on ice. 125 μL each primer:templatemixture was added to a 1 mL extension reaction containing PrimerExtension Buffer, 0.125 U/μL KOD XL DNA Polymerase, and 0.5 mM eachdATP, dCTP, dGTP, and BndUTP, and incubated at 70° C. for 30 minutes.Each 1 mL reaction was split into four 250 aliquots and chilled on ice.Double-stranded product was captured via the template strand biotins byadding 1 mL streptavidin-coated magnetic beads (MagnaBind-Streptavidin,Pierce, 5 mg/mL in 1M NaCl+0.05% TWEEN-20) to each 250 μL aliquot andincubating at 25° C. for 60 minutes with mixing. Beads were washed threetimes with 0.5 mL SB17T Buffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mMKCl, 5 mM MgCl₂, 1 mM EDTA, 0.05% TWEEN-20). The aptamer strand waseluted from the beads with 1 mL 20 mM NaOH, neutralized with 0.25 mL 80mM HCl, and buffered with 10 μL 1 M HEPES, pH 7.5. Candidate mixtureswere concentrated with a Centricon-30 to approximately 0.2 mL, andquantified by UV absorbance spectroscopy.

B. Preparation of Target Proteins

Untagged target proteins were biotinylated by covalent coupling ofNHS-PEO4-biotin (Pierce) to lysines residues. Proteins (300 pmol in 50μL) were exchanged into SB17T with a Sephadex G-25 microspin column.NHS-PEO4-biotin was added to 1.5 mM and the reaction was incubated at 4°C. for 16 hours. Unreacted NHS-PEO4-biotin was removed with a SephadexG-25 microspin column.

C. Aptamer Selection with Slow Off-Rate Enrichment Process andPhotocrosslinking

Selections were performed separately with each candidate mixture,comparing binding between samples with target protein (signal S) andsamples without target protein (background B). The first three roundswere performed with selection for affinity (no photocrosslinking); thesecond and third included slow off-rate enrichment process. Rounds fourthrough eight included both slow off-rate enrichment process andphotocrosslinking.

For each sample, a 90 μL DNA mixture was prepared in SB17T with 10-20pmoles candidate mixture (100 pmoles in the first round) and 100 pmolesreverse primer. Samples were heated to 95° C. for 3 minutes and cooledto 37° C. at a rate of 0.1 C/second. Samples were combined with 10 μLprotein competitor mixture (0.1% HSA, 10 μM casein, and 10 μMprothrombin in SB17T), added to 0.5 mg SA beads (pre-washed twice with20 mM NaOH and once with SB17T), and incubated at 37° C. for 5 minuteswith mixing. Beads were removed by magnetic separation.

Binding reactions were performed by adding 10 μL target protein (0.5 μMin SB17T) or SB17T to 40 μL DNA mixture and incubating at 37° C. for 30minutes.

When slow off-rate enrichment process was employed, samples were diluted20× by adding 950 μL SB17T (preheated to 37° C.), and incubated at 37°C. for 30 minutes prior to capturing complexes.

Complexes were captured on SA beads via protein biotins by adding 0.25mg MyOne-SA beads (Invitrogen) and incubating at 37° C. for 15 minuteswith mixing. Free DNA was removed by washing the beads five times withSB17T. Unless indicated, all washes were performed by resuspending thebeads in 100 μL wash solution, mixing for 30 seconds at 25° C.,separating the beads with a magnet, and removing the wash solution. Theaptamer strand was eluted from the beads by adding 85 μL 20 mM NaOH, andincubating at 37° C. for 1 minute with mixing. 80 μL aptamer eluate wastransferred to a new tube after magnetic separation, neutralized with 20μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl, pH 7.5.

When photo-selection was employed, the 50 μL binding reactions, (or 1 mLbinding reactions after optional slow off-rate enrichment process bydilution) were irradiated from above with a high-pressure mercury lamp(Optical Associates, Inc. model 0131-0003-01, 500 W, with 310 nm mirrorset). Candidate mixtures possessing a BrdU chromophore were irradiatedfor 37 seconds, those possessing an ANA chromophore were irradiated for60 seconds, and those possessing an AQ or psoralen chromophore wereirradiated for 10 minutes. An additional filter (5 mm plate glass) wasused for the ANA, AQ and psoralen chromophores to eliminate unnecessary,but potentially damaging wavelengths below 320 nm. Complexes werecaptured as above, and non-crosslinked DNA was removed by washing thebeads once with 4 M guanidine-HCl+0.05% TWEEN-20 at 50° C. for 10minutes, once with 20 mM NaOH at 25° C. for 2 minutes, twice with SB17T,and once with 16 mM NaCl. Crosslinked DNA was not removed from the beadsurface for the amplification steps.

D. Aptamer Amplification and Purification

Selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA wasadded to 12 μL QPCR Mix (5×KOD DNA Polymerase Buffer, 25 mM MgCl₂, 10 μMforward PCR primer, 10 μM biotinylated reverse PCR primer, 5×SYBR GreenI, 0.125 U/μL KOD XL DNA Polymerase, and 1 mM each dATP, dCTP, dGTP, anddTTP) and thermal cycled in an a Bio-Rad MyIQ QPCR instrument with thefollowing protocol: 1 cycle of 99.9° C., 15 sec, 55° C., 10 sec, 68° C.,30 min, 30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.Quantification was done with the instrument software and the number ofcopies of DNA selected with and without target protein were compared todetermine signal/background ratios.

When photo-selection was employed, a cDNA copy of the selected DNA wasprepared by primer extension on the bead surface. Washed beads wereresuspended in 20 μL cDNA extension mix (Primer Extension Buffercontaining 5 μM reverse PCR primer, 0.5 mM each dATP, dCTP, dGTP, anddTTP, and 0.125 U/μL KOD XL DNA Polymerase) and incubated at 68° C. for30 minutes with mixing. The beads were washed 3 times with SB17T, andthe aptamer strand was eluted by from the beads by adding 85 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. 80 μL aptamereluate was transferred to a new tube after magnetic separation,neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl,pH 7.5. The cDNA was amplified and quantified by QPCR as above for the30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.

Following amplification, the PCR product was captured on SA beads viathe biotinylated antisense strand. 1.25 mL SA beads (10 mg/mL) werewashed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL SB17T, resuspendedin 1.25 mL 3 M NaCl+0.05% Tween, and stored at 4° C. 25 μL SA beads (10mg/mL in 3 M NaClT) were added to 50 μL double-stranded QPCR product andincubated at 25° C. for 5 minutes with mixing. The beads were washedonce with SB17T, and the “sense” strand was eluted from the beads byadding 200 μL 20 mM NaOH, and incubating at 37° C. for 1 minute withmixing. The eluted strand was discarded and the beads were washed 3times with SB17T and once with 16 mM NaCl.

Aptamer sense strand was prepared with the appropriate chromophore byprimer extension from the immobilized antisense strand. The beads wereresuspended in 20 μL primer extension reaction mixture (1× PrimerExtension Buffer, 1.5 mM MgCl₂, 5 μM forward primer with appropriate 5′chromophore, 0.5 mM each dATP, dCTP, dGTP, and BndUTP, and 0.125 U/μLKOD XL DNA Polymerase) and incubated at 68° C. for 30 minutes withmixing. The beads were washed 3 times with SB17T, and the aptamer strandwas eluted from the beads by adding 85 μL 20 mM NaOH, and incubating at37° C. for 1 minute with mixing. 80 μL aptamer eluate was transferred toa new tube after magnetic separation, neutralized with 20 μL 80 mM HCl,and buffered with 5 μL 0.1 M HEPES, pH 7.5.

E. Selection Stringency and Feedback

Target protein was adjusted at each round as described in Example 1.After each round of selection, the convergence state of the enrichedpool was determined as described in Example 1.

F. Equilibrium Binding Constants of Enriched Libraries

The binding affinity was determined as described in Example 1 above, butwith SA capture beads. The following table, Table 2, summarizes theequilibrium binding constants (K_(d)) obtained using the photoSELEXprotocol with slow off-rate enrichment process.

TABLE 1 Equilibrium binding constants (K_(d)) of the enriched librariesselected with different modified nucleotides, reported in units ofmolarity. NT = not tested. Target Protein dTTP BndUTP iBudUTP TrpdUTP4-1BB  >1.0 × 10⁻⁷ 5.6. × 10⁻⁹ >1.0. × 10⁻⁷  3.9. × 10⁻⁹ B7 >1.0. × 10⁻⁷1.1. × 10⁻⁸ NT 7.2. × 10⁻⁹ B7-2 >1.0. × 10⁻⁷ NT >1.0. × 10⁻⁷  5.7. ×10⁻⁹ CTLA-4 >1.0. × 10⁻⁷ NT NT 1.4. × 10⁻⁹ E-Selectin >1.0. × 10⁻⁷ >1.0.× 10⁻⁷  >1.0. × 10⁻⁷  1.9. × 10⁻⁹ Fractal- NT >1.0. × 10⁻⁷  NT  5.1. ×10⁻¹¹ kine GA733-1  8.9. × 10⁻⁹ 2.8. × 10⁻⁹ 4.7. × 10⁻⁹  4.5. × 10⁻¹⁰protein Gp130 >1.0. × 10⁻⁷ 5.9. × 10⁻⁹ 2.2. × 10⁻⁸ 1.2. × 10⁻⁹HMG-1 >1.0. × 10⁻⁷ NT 2.2. × 10⁻⁸ 4.9. × 10⁻⁹ IR >1.0. × 10⁻⁷ 1.9. ×10⁻⁹ 1.2. × 10⁻⁸  2.2. × 10⁻¹⁰ OPG  3.7. × 10⁻⁸ 4.6. × 10⁻⁹ 9.5. × 10⁻⁹ 1.7. × 10⁻¹⁰ PAI-1 >1.0. × 10⁻⁷  3.7. × 10⁻¹⁰  9.1. × 10⁻¹⁰  4.3. ×10⁻¹⁰ P- >1.0. × 10⁻⁷ 3.5. × 10⁻⁹ 5.2. × 10⁻⁹ 2.7. × 10⁻⁹ CadherinsLeptin R >1.0. × 10⁻⁷ 2.3. × 10⁻⁹ NT  4.6. × 10⁻¹⁰

G. Crosslink Activity Assay

The crosslink yield of enriched libraries was determined by measuringthe percent of DNA crosslinked to protein under conditions of saturatingprotein and light. Radiolabeled DNA (50 pM) was mixed with reverseprimer (16 nM) in SB17T, heated to 95° C. for 3 minutes, and cooled to37° C. at 0.1° C./second. Target protein was added to the DNA mix to afinal concentration of 10 nM and incubated at 37° C. for 30 minutes.Control samples with no protein were simultaneously prepared. Sampleswere crosslinked with the chromophore-specific conditions describedabove, but with a saturating dose (6 minutes for BrdU, 10 minutes forANA, and 30 minutes for AQ and Psor). Samples were analyzed bydenaturing PAGE, FIG. 6, and quantified and the results are tabulated inTable 3.

TABLE 3 Crosslink yields of the enriched libraries selected withdifferent chromophores, reported in units of percent of total DNAcrosslinked to protein. Measurements were not made on libraries thatfailed to converge (indicated with an x). Target Protein BrdU AQ ANAPsor β-catenin 15  9 8 1 bFGF 4 9 15 4 CMP-SAS x 3 5 2 Endostatin 2 1 183 IL-6 0 5 9 Myeloperoxidase 4 13 9 8 SDF-1β 8 10 17 x TIMP-1 1 4 2 xVEGF 1 1 4 x vWF 2 2 7 x

Example 3. Generation of Slow Off-Rate Aptamers Using a Slow Off-RateEnrichment Process with a Competitor

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP were preparedby polymerase extension of a primer annealed to a biotinylated templatefor 94 protein targets. 55 nmol forward primer (with 5′ ANA chromophore)and 55 nmol template were combined in 0.5 mL Primer Extension Buffer(120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6 mM (NH₄)₂SO₄, 7 mM MgSO₄, 0.1mg/mL BSA, 0.1% Triton X-100), heated to 95° C. for 5 minutes, 70° C.for 5 minutes, 48° C. for 5 minutes, and cooled on ice. Theprimer:template mixture was added to a 5.5 mL extension reactioncontaining Primer Extension Buffer, 0.125 U/μL KOD XL DNA Polymerase,and 0.5 mM each dATP, dCTP, dGTP, and BndUTP, and incubated at 70° C.for 60 minutes. After completion of the extension reaction, the solutionwas chilled on ice. Double-stranded product was captured via thetemplate strand biotins by adding 25 mL streptavidin-coated magneticbeads (MagnaBind-Streptavidin, Pierce, 5 mg/mL in 1 M NaCl+0.05%TWEEN-20) to the primer extension product and incubating 25° C. for 15minutes with rotating. Beads were washed three times with 40 mL SB17TBuffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mMEDTA, 0.05% TWEEN-20). The aptamer strand was eluted from the beads with35.2 mL 20 mM NaOH for 5 minutes with shaking. The eluted strand wasneutralized with 8.8 mL 80 mM HCl, and buffered with 400 μL 1 M HEPES,pH 7.3. Candidate mixtures were concentrated with a Centricon-30 toapproximately 0.7 mL, and quantified by UV absorbance spectroscopy.

B. Preparation of Target Proteins

Untagged target proteins were biotinylated as described in Example 2.

C. Aptamer Selection with Slow Off-Rate Enrichment Process andPhotocrosslinking

Selections were performed separately as described in Example 2, with theaddition of 10 mM dextran sulfate as a competitor for aptamer rebindingduring the slow off-rate enrichment process in rounds six through nine.

The slow off-rate enrichment process was employed in three differentways. In rounds two and three, samples were diluted 20× by adding 950 μLSB17T (preheated to 37° C.), and incubated at 37° C. for 30 minutesprior to capturing complexes. In rounds four and five, samples werediluted 20× by adding 950 μL SB17T (preheated to 37° C.), and incubatedat 37° C. for 30 minutes prior to crosslinking. In rounds six and seven,samples were diluted 20× by adding 950 μL SB17T (preheated to 37° C.).50 μL of each diluted sample was diluted again by transferring to 950 μLSB17T+10 mM 5000K dextran sulfate (preheated to 37° C.) to give anoverall 400× dilution, and incubated at 37° C. for 60 minutes prior tocrosslinking. In rounds eight and nine, samples were diluted 20× byadding 950 μL SB17T (preheated to 37° C.), and 50 of each sample wasdiluted again by transferring to 950 μL SB17T (preheated to 37° C.) togive 400× dilution. Finally, 50 μL of each 400× diluted sample wasdiluted again by transferring to 950 μL SB17T+10 mM 5000K dextransulfate (preheated to 37° C.) to give an overall 8000× dilution, andincubated at 37° C. for 60 minutes prior to crosslinking. Complexes werecaptured and washed as described in Example 2. When photo-crosslinkingwas employed, the 1 mL binding reactions after the slow off-rateenrichment process were irradiated from above with an array of 470 nmLEDs for 60 seconds prior to complex capture as in Example 2.

D. Aptamer Amplification and Purification

Amplification and purification were performed as in Example 2.

E. Selection Stringency and Feedback

Target protein was adjusted at each round as described in Example 1,except in rounds six and eight. In order to maximize signal after theselarge dilutions, the target protein was increased to 100 nM for roundssix and eight. After each round of selection, the convergence state ofthe enriched pool was determined as described in Example 1.

F. Dissociation Rate Constant Determination Protocol.

The rate constant for aptamer:protein complex dissociation (koff) wasdetermined for each aptamer by measuring the fraction of pre-formedaptamer:protein complexes that remain bound after dilution as a functionof time. Radiolabeled aptamer (50 pM) was equilibrated in SB17T-0.002(SB17T with TWEEN-20 reduced to 0.002%) at 37° C. with protein at aconcentration 10× greater than the measured K_(d) value. Samples werediluted 100× with SB17T-0.002 at 37° C. and aliquots were removed atvarious time points and partitioned to separate free aptamer fromprotein:aptamer complexes. Partitioning was accomplished by addingZORBAX resin (Agilent) to the sample, capturing complexes on the resin,passing the sample through a DuraPore membrane under vacuum, and washingthe resin with SB17T-0.002. For proteins not efficiently captured withZORBAX resin, the assay was performed with biotinylated protein in SB17Tand partitioning was accomplished by capturing complexes with SA beads.The amount of complex remaining at each time point was determined byquantifying the radiolabeled aptamer on the resin with a FUJI FLA-3000phosphorimager. The fraction of complex was plotted as a function oftime and the dissociation rate constant (koff) and dissociationhalf-life value (t_(1/2)) was determined by fitting the data to ananalytic expression for bimolecular dissociation kinetics usingnon-linear regression.

G. Kinetic Properties of Some Aptamers

The following table, Table 4, summarizes the dissociation half-lifevalues (t_(1/2)) obtained for aptamers selected against 10 targets usingthis protocol.

TABLE 4 Dissociation half-life values (t_(1/2)) of aptamers using thecompetitor slow off-rate enrichment step protocol. Target Proteint_(1/2) (min) bFGF R 66 C3 164 catalase 58 FGF-17 91 group IBphospholipase A2 40 HB-EGF 49 HCC-4 143 IL-6 sRα 114 SAP 186

Example 4. The Slow Off-Rate Enrichment Process Increases theDissociation Half-Life of Selected Aptamers

Dissociation half-life values (t_(1/2)) were measured and plotted for 65aptamers that were selected by either the affinity SELEX methoddescribed in Example 1 or photo SELEX methods described in U.S. Pat. No.6,458,539, entitled “Photoselection of Nucleic Acid Ligands” without aslow off-rate enrichment process (FIG. 3A). t_(1/2) values were alsomeasured and plotted for 72 aptamers that were selected by the slowoff-rate enrichment process described in Example 2 with a slow off-rateenrichment process by dilution or dilution with competitor (FIG. 3B).The average t_(1/2) value for aptamers using the modified nucleotides5-(N-benzylcarboxyamide)-dUTP (BndUTP), 5-(N-isobutylcarboxyamide)-dUTP(iBudUTP), or 5-(N-tryptaminocarboxyamide)-dUTP (TrpdUTP) selected inthe absence of a slow off-rate enrichment process was 20 minutes withsome aptamers having a t_(1/2) value of up to one hour. This issubstantially longer than what has been previously described withnatural bases or other modified nucleotides. The average for aptamersselected with a slow off-rate enrichment process was over 85 minutes,with some aptamers having a t_(1/2) value in excess of four hours.

Example 5. Generation of Aptamers from a NapdU Random Library

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and NapdUTP wereprepared as described in Example 3 but without the 5′-ANA photoreactivegroup.

B. Immobilization of Target Proteins Target proteins contained a (His)₆tag and were captured with Talon beads as described in Example 1.

C. Aptamer Selection with Slow Off-Rate Enrichment Process

Aptamer selection was performed as described in Example 3, but withoutphotocrosslinking.

D. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

E. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

F. Aptamer Properties

The equilibrium binding constant (K_(d)) of four aptamers from thisselection are listed in Table 5.

TABLE 5 Equilibrium binding constants (Kd) of NpdUTP aptamers TargetProtein K_(d) (M) bFGF 1.1. × 10⁻⁹  Endostatin 2.0. × 10⁻¹⁰ TIMP-3 1.5.× 10⁻¹⁰ VEGF 7.2. × 10⁻¹⁰

Example 6. Generation of Slow-Off-Rate Aptamers for a Peptide TargetUsing a Slow Off-Rate Enrichment Process with a Competitor

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BndUTP were preparedby polymerase extension of a primer with a 5′ ANA chromophore andpurified as described in Example 3.

B. Aptamer Selection with Slow Off-Rate Enrichment Process andPhotocrosslinking

Aptamer selection was performed as described in Example 3 with the 29amino acid biotinylated target peptide SMAP29 (Sheep MyeloidAntibacterial Peptide MAP-29, Anaspec).

C. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

D. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

E. Aptamer Properties

The equilibrium binding constant (K_(d)) of an aptamer from thisselection was 1.2.×10⁻⁸M (measured according to the protocol describedin Example 1). The dissociation half-life (t_(1/2)) of this aptamer was69 minutes (measured according to the protocol described in Example 3).Results are shown in FIG. 12A and FIG. 12B.

Example 7. Protein Measurements in Test Samples were Enabled by Aptamerswith Slow Off-Rates

A. Preparation of Aptamer/Primer Mixtures and Test Samples

Aptamers with a biotin Cy3 detection label (4 nM each) were mixed with a3× excess of capture probe (oligonucleotide complementary to the 3′fixed region of the aptamer containing a biotin tag and photocleavableelement) in 1×SB17T and heated at 95° C. for 4 minutes, then 37° C. for13 minutes, and diluted 1:4 in 1×SB17T. 55 uL of aptamer/primer mix wasadded to a microtiter plate (Hybaid #AB-0407) and sealed with foil. Testsamples were prepared in a microtiter plate by mixing knownconcentrations of protein analytes in SB17T and diluting serially withSB17T.

B. Sample Equilibration

55 uL of aptamer/primer mix was added to 55 uL of test sample andincubated at 37° C. for 15 minutes in a foil-sealed microtiter plate.The final concentration of each aptamer in the equilibration mixture was0.5 nM. After equilibration, all subsequent steps of this method wereperformed at room temperature unless otherwise noted.

C. Aptamer Capture and Free Protein Removal

A DuraPore filtration plate (Millipore HV cat #MAHVN4550) was washedonce with 100 uL 1×SB17T by vacuum filtration. 133.3 uL 7.5%Streptavidin-agarose resin (Pierce) was added to each well and washedtwice with 200 uL 1×SB17T. 100 uL of equilibrated samples wastransferred to the Durapore plate containing the Streptavidin-agaroseresin and incubated on a thermomixer (Eppendorf) at 800 rpm for 5minutes. The resin was washed once with 200 uL 1×SB17T+100 uM biotin andonce with 200 uL 1×SB17T.

D. Protein Tagging with Biotin

100 uL of 1.2 mM NHS-PEO4-biotin in SB17T, prepared immediately beforeuse, was added to the resin with captured aptamer and aptamer:proteincomplexes and incubated on a thermomixer at 800 rpm for 20 minutes. Theresin was washed five times with 200 uL 1×SB17T by vacuum filtration.

E. Slow Off-Rate Enrichment Process & Photocleavage

The drip director was removed from underside of the DuraPore plate andthe plate was placed over a 1 mL microtiter collection plate. The resinwas washed once with 200 uL 1×SB17T by centrifugation at 1000×g for 30sec. 80 uL of 1×SB17T+10 mM dextran sulfate was added to the resin andirradiated with a BlackRay Mercury Lamp on a thermomixer at 800 rpm for10 minutes. The DuraPore plate was transferred to a new 1 mL deepwellplate and centrifuged at 1000×g for 30 seconds to collect thephotocleaved aptamer and protein:aptamer complexes.

F. Protein Capture and Free Aptamer Removal

50 uL of MyOne-streptavidin Cl paramagnetic beads (Invitrogen) (10 mg/mLin 1×SB17T) was added to a microtiter plate. The beads were separatedwith a magnet for 60 seconds and the supernatant was removed. 225 uL ofphotocleavage mixture was added to the beads and mixed for 5 minutes.The beads were washed four times with 200 uL 1×SB17T by separating themagnetic beads and replacing the wash buffer. The final wash buffer wasremoved.

G. Aptamer Elution

100 uL Sodium Phosphate Elution Buffer (10 mM Na₂HPO₄, pH 11) was addedto the beads and mixed for 5 minutes. 90 uL of eluate was transferred toa microtiter plate and neutralized with 10 uL Sodium PhosphateNeutralization Buffer (10 mM NaH₂PO₄, pH 5).

H. Aptamer Hybridization to Microarrays

DNA arrays were prepared with oligonucleotide capture probes comprisedof the complementary sequence of the variable region of each aptamerimmobilized on a custom microscope slide support. Multiple arrays(subarrays) existed on each slide, and subarrays were physicallyseparated by affixing a gasket (Grace) for sample application. Arrayswere pretreated with 100 uL Blocking Buffer and incubated for 15 minutesat 65° C. on a thermomixer. 30 uL of high salt Hybridization Buffer wasadded to 90 uL of neutralized aptamer eluate in a microtiter plate,incubated at 95° C. for 5 minutes in a thermalcycler, and cooled to 65°C. at 0.1° C./second. Blocking Buffer was removed from the arrays and110 uL of aptamer sample was added to the arrays and incubate in a humidchamber at 65° C. for 20 hours.

I. Array Washing

Aptamer sample was removed from the arrays, and the arrays were washedonce with 200 uL of sodium phosphate Tween-20 wash buffer at 65° C.,with the gasket in place, and three times with 25 mL sodium phosphate,Tween-20 wash buffer at 65° C. in a pap jar with the gasket removed.Arrays were dried with a nitrogen gun.

J. Quantitate Signal on Arrays

Array slides were scanned on a TECAN LS300 in an appropriate channel forCy3 detection and Cy3 signal on each array feature was quantified.

Results:

Apatmers specific to three different targets (bFGF, VEGF, andMyeloperoxidase) were produced using traditional SELEX methods andmaterials. A second set of aptamers specific to the same set of targetswere made using 5-position modified nucleotides and selected for veryslow off-rates for their respective targets. Aptamers made in thetraditional process had measured off rates on the order of less than 5minutes. Aptamers made with the modified nucleotides and using slowoff-rate enrichment process during selection had off rates of greaterthan 20 minutes. Two sets of aptamers were made for each target by thetwo different methods for a total of 4 different aptamer populations foreach target. The ability of these aptamer populations to measure analyteconcentrations in test samples was evaluated as described above over arange of target concentrations. Relative signal from the DNA chipdetection was plotted against the input target concentration. See FIGS.11A to 11C. The response curve of the traditional aptamers was very flatand the sensitivity of the detection was fairly low. The sensitivity ofdetection of the respective targets with the slow off-rate aptamers wasexcellent. The data supports the need to use the slow off-rate aptamersfor maximum analytic performance.

Example 8. Generation of High Affinity BndU Aptamers to Human Thrombin

A. Preparation of Candidate Mixture

A candidate mixture containing dATP, dCTP, dGTP, and BndUTP was preparedby polymerase extension of a primer with a 5′ ANA chromophore andpurified as described in Example 3.

B. Preparation of Target Protein

Human thrombin was tagged with biotin as describe in Example 2.

C. Aptamer Selection with Slow Off-Rate Enrichment and Photocrosslinking

Aptamer selection was performed as described in Example 3 withbiotinylated human thrombin as the target.

D. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

E. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

F. Aptamer Properties

The equilibrium binding constant (K_(d)) of aptamer 2336-17 from thisselection with a modified BndU was 4.4×10¹¹M (measured according to theprotocol described in Example 1) as demonstrated in FIG. 15. In the art,single-stranded DNA aptamers to human thrombin were selected from alibrary comprised of natural dA, dC, dG, and dT nucleotides (Bock, etal., Selection of Single-Stranded DNA Molecules that Bind and InhibitHuman Thrombin, Nature 1992 355: 564-566). The binding affinities of theaptamers had K_(d) values ranging from 2.5×10⁻⁸M to 2.0×10⁻⁷ M. Using asimilar protocol with a library comprised of natural dA, dC, dG, andmodified 5-(1-pentynyl)-dUTP, aptamers were selected with K_(d) valuesranging from 4×10⁻⁷ M to 1×10⁻⁶ M (Latham, et al., The Application of aModified Nucleotide in Aptamer Selection: Novel Thrombin AptamersContaining 5-(1-Pentynyl)-2′-Deoxyuridine, Nucleic Acid Research 199422(14): 2817-2822).

A number of patents, patent application publications, and scientificpublications are cited throughout and/or listed at the end of thedescription. Each of these is incorporated herein by reference in theirentirety. Likewise, all publications mentioned in an incorporatedpublication are incorporated by reference in their entirety.

Examples in cited publications and limitations related therewith areintended to be illustrative and not exclusive. Other limitations of thecited publications will become apparent to those of skill in the artupon a reading of the specification and a study of the drawings.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively.

1.-134. (canceled)
 135. A compound comprising a 5-position modifiedpyrimidine, wherein the group attached via a linker to the 5-position isa hydrophobic group selected from the group consisting of naphthyl,napthylmethyl, phenyl, benzyl, and indole.
 136. The compound of claim135, wherein the hydrophobic group is selected from benzyl, indole andnaphthyl.
 137. The compound of claim 135, wherein the 5-positionmodified pyrimidine is a 5-position modified uridine or cytosine. 138.The compound of claim 135, wherein the 5-position modified pyrimidine isa nucleotide in an oligonucleotide.
 139. The compound of claim 138,wherein the oligonucleotide is an aptamer.
 140. The compound of claim139, wherein the aptamer has binding affinity for a target molecule.141. The compound of claim 140, wherein the target molecule is a proteinor peptide.
 142. The compound of claim 135, wherein the linker isselected from the group of linkers consisting of: —(CH₂)_(n)—NH—C(═O)—;—(CH₂)_(n)—C(═O)—; —(CH₂)_(n)—O—C(═O)—; —(CH₂)_(n)—NH—C(═O)—;—(CH₂)_(n)—O—C(═O)—NH—; —(CH₂)_(n)—S(═O)—; —(CH₂)_(n)—S(═O)₂—; and—(CH₂)_(n)—C(═O)—NH—, where n is 0-3 and wherein the right side of thelinker is bonded to the 5′ position of the pyrimidine and the left sideof the linker is bonded to the hydrophobic group.
 143. The compound ofclaim 142, wherein the linker is —(CH₂)_(n)—NH—C(═O)—, where n is 0-3.144. The compound of claim 143, wherein the 5-position modifiedpyrimidine is a 5-position modified cytosine or uridine.
 145. Thecompound of claim 144, wherein the group and linker attached to the5-position is


146. The compound of claim 144, wherein the group and linker attached tothe 5-position is


147. The compound of claim 144, wherein the linker is —C(═O)—NH—(CH₂)₃—and the group attached via the linker to the 5-position is phenyl. 148.The compound of claim 144, wherein the linker is —C(═O)—NH—(CH₂)— andthe group attached via the linker to the 5-position is

where

denotes the point of attachment of the group to —CH₂—.
 149. The compoundof claim 144, wherein the linker is —C(═O)—NH—(CH₂)— and the groupattached via the linker to the 5-position is


150. The compound of claim 135, wherein the 5-position modifiedpyrimidine of the aptamer improves the binding affinity for the targetmolecule compared to the aptamer without the 5-position modifiedpyrimidine.